|
|
| Line 7,879: |
Line 7,879: |
| | <body id="bootstrap-overrides"> | | <body id="bootstrap-overrides"> |
| | <h3>Lab Notebook</h3> | | <h3>Lab Notebook</h3> |
| − | <p><br></p>
| + | |
| | <p class ="main"> | | <p class ="main"> |
| | <img src="https://static.igem.org/mediawiki/2016/9/97/Denver_Biolabs_RFP.jpg" alt="Comp cell test" style="max-width:100%; height:auto;" class="img-responsive center-block transp nuoli"> | | <img src="https://static.igem.org/mediawiki/2016/9/97/Denver_Biolabs_RFP.jpg" alt="Comp cell test" style="max-width:100%; height:auto;" class="img-responsive center-block transp nuoli"> |
| | <!-- INSERT PAGE CONTENT HERE!! --> | | <!-- INSERT PAGE CONTENT HERE!! --> |
| − | | + | <iframe width='100%' height='1500' src="https://docs.google.com/document/d/1g6an7zJoXDKNMhRi_GKjh8ymUUPzepS5awtHSfGt6Y0/pub?embedded=true"></iframe> |
| − | <!-- | + | |
| − | /* Font Definitions */
| + | |
| − | @font-face
| + | |
| − | {font-family:Arial;
| + | |
| − | panose-1:2 11 6 4 2 2 2 2 2 4;}
| + | |
| − | @font-face
| + | |
| − | {font-family:"Cambria Math";
| + | |
| − | panose-1:2 4 5 3 5 4 6 3 2 4;}
| + | |
| − | @font-face
| + | |
| − | {font-family:Calibri;
| + | |
| − | panose-1:2 15 5 2 2 2 4 3 2 4;}
| + | |
| − | /* Style Definitions */
| + | |
| − | p.MsoNormal, li.MsoNormal, div.MsoNormal
| + | |
| − | {margin:0in;
| + | |
| − | margin-bottom:.0001pt;
| + | |
| − | font-size:12.0pt;
| + | |
| − | font-family:Calibri;}
| + | |
| − | p
| + | |
| − | {margin-right:0in;
| + | |
| − | margin-left:0in;
| + | |
| − | font-size:12.0pt;
| + | |
| − | font-family:"Times New Roman";}
| + | |
| − | .MsoChpDefault
| + | |
| − | {font-family:Calibri;}
| + | |
| − | @page WordSection1
| + | |
| − | {size:8.5in 11.0in;
| + | |
| − | margin:1.0in 1.0in 1.0in 1.0in;}
| + | |
| − | div.WordSection1
| + | |
| − | {page:WordSection1;}
| + | |
| − | /* List Definitions */
| + | |
| − | ol
| + | |
| − | {margin-bottom:0in;}
| + | |
| − | ul
| + | |
| − | {margin-bottom:0in;}
| + | |
| − | -->
| + | |
| − | <article class="article">
| + | |
| − | | + | |
| − | <div class=WordSection1>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:red'>Updated
| + | |
| − | through 10/10</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:red'>Red
| + | |
| − | text needs double checking, blue text is extraneous comments</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal style='margin-bottom:12.0pt'><span style='font-family:"Times New Roman"'> </span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'>8/1/16
| + | |
| − | - Ryan</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'>LB
| + | |
| − | agar plates - made media for plates</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'> Dissolved
| + | |
| − | 20 g LB broth/agar in 350 mL DI H2O</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'> Stirred
| + | |
| − | with magnetic stir bar until dissolved</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'> Added
| + | |
| − | 150 mL DI H2O to bring total volume to 500 mL (0.5 L)</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'> Divided
| + | |
| − | solution into 2 500 mL flasks</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'> Autoclaved
| + | |
| − | on liquid cycle for 20 minutes</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'> Allowed
| + | |
| − | solution to cool to 55 degrees C</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'> Added
| + | |
| − | 500 </span><span style='font-size:11.0pt;font-family:"Cambria Math";color:black'>𝜇</span><span
| + | |
| − | style='font-size:11.0pt;font-family:Arial;color:black'>L of Chlor to solution</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'> Labelled
| + | |
| − | plates (2 parallel purple lines on side to indicate Chlor plates)</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'> Poured
| + | |
| − | plates and allowed to set.</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-family:"Times New Roman"'> </span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'>8/4/16
| + | |
| − | Isaac, Ingrid</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'>LB
| + | |
| − | plates (without antibiotics and with Chlor)</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'> Dissolved
| + | |
| − | 8 g LB agar (BioBasic) in 200 mL DI H2O (Rocky Mountain Reagent)</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'> Stirred
| + | |
| − | with magnetic stir bar until dissolved</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'> Autoclaved
| + | |
| − | on liquid cycle with stir bar for 30 minutes (flask in back of autoclave</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'> Liquid
| + | |
| − | boiled over from 200 mL to</span><span style='font-size:11.0pt;font-family:
| + | |
| − | Arial;color:red'> *** </span><span style='font-size:11.0pt;font-family:Arial;
| + | |
| − | color:black'>mL)</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'> Autoclaved
| + | |
| − | liquid without stir bar for 30 minutes (with flask in center and a bit of water
| + | |
| − | in the rack to create steam and prevent boiling over)</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'> Poured
| + | |
| − | 2 plates</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'> Added
| + | |
| − | </span><span style='font-size:11.0pt;font-family:Arial;color:red'>***</span><span
| + | |
| − | style='font-size:11.0pt;font-family:Arial;color:black'> </span><span
| + | |
| − | style='font-size:11.0pt;font-family:"Cambria Math";color:black'>𝜇</span><span
| + | |
| − | style='font-size:11.0pt;font-family:Arial;color:black'>L (@ 25 </span><span
| + | |
| − | style='font-size:11.0pt;font-family:"Cambria Math";color:black'>𝜇</span><span
| + | |
| − | style='font-size:11.0pt;font-family:Arial;color:black'>g/mL) of Chlor to
| + | |
| − | remaining solution</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'> Labelled
| + | |
| − | Chlor plates with 2 parallel purple lines</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'> Poured
| + | |
| − | </span><span style='font-size:11.0pt;font-family:Arial;color:red'>**</span><span
| + | |
| − | style='font-size:11.0pt;font-family:Arial;color:black'> plates with LB agar +
| + | |
| − | Chlor</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-family:"Times New Roman"'> </span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'>Transformed
| + | |
| − | e. coli competent cell from NEB with competent cell kit by iGEM team</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'> Spun
| + | |
| − | down DNA tubes (50 pg/</span><span style='font-size:11.0pt;font-family:"Cambria Math";
| + | |
| − | color:black'>𝜇</span><span style='font-size:11.0pt;font-family:Arial;
| + | |
| − | color:black'>L and 0.5 pg/</span><span style='font-size:11.0pt;font-family:
| + | |
| − | "Cambria Math";color:black'>𝜇</span><span style='font-size:11.0pt;
| + | |
| − | font-family:Arial;color:black'>L) for 30 seconds at 9000 rpm to collect cells
| + | |
| − | in bottom of tube</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'> Thawed
| + | |
| − | competent e. coli cells from NEB on ice for </span><span style='font-size:11.0pt;
| + | |
| − | font-family:Arial;color:red'>15</span><span style='font-size:11.0pt;font-family:
| + | |
| − | Arial;color:black'> minutes</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'> Pipetted
| + | |
| − | 1 </span><span style='font-size:11.0pt;font-family:"Cambria Math";color:black'>𝜇</span><span
| + | |
| − | style='font-size:11.0pt;font-family:Arial;color:black'>L of DNA into 2
| + | |
| − | appropriately labelled microcentrifuge tubes (50 and 0.5)</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'> Pipetted
| + | |
| − | 1 </span><span style='font-size:11.0pt;font-family:"Cambria Math";color:black'>𝜇</span><span
| + | |
| − | style='font-size:11.0pt;font-family:Arial;color:black'>L of BBa_K775000 into
| + | |
| − | appropriately labelled tube (BBa 5000)</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'> Added
| + | |
| − | 50 </span><span style='font-size:11.0pt;font-family:"Cambria Math";color:black'>𝜇</span><span
| + | |
| − | style='font-size:11.0pt;font-family:Arial;color:black'>L competent e. coli
| + | |
| − | cells into each of the 3 tubes; mixed tubes by flicking each gently with finger</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'> Incubated
| + | |
| − | on ice for 30 minutes</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'> Heat
| + | |
| − | shocked cells in 42 degree C water bath for 1 minute; immediately put tubes
| + | |
| − | back on ice after water bath</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'> Added
| + | |
| − | 200 </span><span style='font-size:11.0pt;font-family:"Cambria Math";color:black'>𝜇</span><span
| + | |
| − | style='font-size:11.0pt;font-family:Arial;color:black'>L of SOC media to each
| + | |
| − | tube; incubate tubes at 39 degrees C on shaking incubator for 2 hours (SOC
| + | |
| − | media added to tubes next to butane burner for more sterile work environment)</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'> Plated
| + | |
| − | BBa_K775000 onto 2 plates at 2 different concentrations</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'> @
| + | |
| − | 20 </span><span style='font-size:11.0pt;font-family:"Cambria Math";color:black'>𝜇</span><span
| + | |
| − | style='font-size:11.0pt;font-family:Arial;color:black'>L per plate</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'> @
| + | |
| − | 200 </span><span style='font-size:11.0pt;font-family:"Cambria Math";color:black'>𝜇</span><span
| + | |
| − | style='font-size:11.0pt;font-family:Arial;color:black'>L per plate</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'> Plated
| + | |
| − | competent cell test solutions (50 and 0.5) on 3 plates each (for 6 plates
| + | |
| − | total)</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'> @
| + | |
| − | 20 </span><span style='font-size:11.0pt;font-family:"Cambria Math";color:black'>𝜇</span><span
| + | |
| − | style='font-size:11.0pt;font-family:Arial;color:black'>L per plate</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'> Placed
| + | |
| − | all plates in 37 degree incubator</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-family:"Times New Roman"'> </span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'>8/6/16
| + | |
| − | - Isaac</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'>Made
| + | |
| − | LB plates to restreak NEB competent cells so we can select a single virile
| + | |
| − | colony to grow up in SOC</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'> Restreaked
| + | |
| − | 3 LB plates, incubated @ 37 degrees C</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-family:"Times New Roman"'> </span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'>Miniprepped
| + | |
| − | overnight cultures of BBa_K775000</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'> Used
| + | |
| − | 3 overnight tubes with 5 mL each to make 6 microcentrifuge tubes with 2 ml each</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'> Used
| + | |
| − | cryo tubes for larger capacity</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'> Centrifuged
| + | |
| − | tubes at 8000 rpm for 2 minutes; decanted supernatant</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'> Paired
| + | |
| − | up tubes to make 3 sets of 2 tubes each</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'> For
| + | |
| − | each set, added 150 </span><span style='font-size:11.0pt;font-family:"Cambria Math";
| + | |
| − | color:black'>𝜇</span><span style='font-size:11.0pt;font-family:Arial;
| + | |
| − | color:black'>L to one tube and 100 </span><span style='font-size:11.0pt;
| + | |
| − | font-family:"Cambria Math";color:black'>𝜇</span><span style='font-size:
| + | |
| − | 11.0pt;font-family:Arial;color:black'>L to the other tube</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'> Vortexed
| + | |
| − | all tubes to resuspend</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'> Poured
| + | |
| − | 150 </span><span style='font-size:11.0pt;font-family:"Cambria Math";color:black'>𝜇</span><span
| + | |
| − | style='font-size:11.0pt;font-family:Arial;color:black'>L from each set into 100
| + | |
| − | </span><span style='font-size:11.0pt;font-family:"Cambria Math";color:black'>𝜇</span><span
| + | |
| − | style='font-size:11.0pt;font-family:Arial;color:black'>L from same set to make
| + | |
| − | 3 tubes with 250 </span><span style='font-size:11.0pt;font-family:"Cambria Math";
| + | |
| − | color:black'>𝜇</span><span style='font-size:11.0pt;font-family:Arial;
| + | |
| − | color:black'>L resuspension solution + bacteria</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'> From
| + | |
| − | there, followed Genejet protocol for 3 250 </span><span style='font-size:11.0pt;
| + | |
| − | font-family:"Cambria Math";color:black'>𝜇</span><span style='font-size:
| + | |
| − | 11.0pt;font-family:Arial;color:black'>L tubes</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'> Protocol
| + | |
| − | Deviation: did not do optional step 7</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'> Protocol
| + | |
| − | Deviation:did not heat elution buffer to 70 degrees </span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'> Protocol
| + | |
| − | Deviation: did not do second elution step</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'> Used
| + | |
| − | 1 tube for restriction digest; put 2 tubes in -20 degree C freezer for later
| + | |
| − | use</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-family:"Times New Roman"'> </span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'>Restriction
| + | |
| − | Digest </span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'> Used
| + | |
| − | SnapGene to create new file (BBa_K775000 +pSBIC3.dna); filed in Google Drive
| + | |
| − | �DNA SnapGene� folder </span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'> In
| + | |
| − | SnapGene, used �Choose Enzymes� tool to make set of available DBL enzymes;
| + | |
| − | saved set as �DBL Enzymes 8 6 2016.txt�; uploaded set to Google Drive �DNA
| + | |
| − | SnapGene� folder</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'> Selected
| + | |
| − | �DBL Enzymes� to evaluate plasmid map</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'> Used
| + | |
| − | �Simulate Agarose Gel� tool to test enzyme options</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'> Selected
| + | |
| − | BglII to cut inside BBa part</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'> Selected
| + | |
| − | NotI to give 3 bands of different sizes</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'>Set
| + | |
| − | up for restriction digest using the following:</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal style='text-indent:.5in'><span style='font-size:11.0pt;
| + | |
| − | font-family:Arial;color:black'>Sample 1: </span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'> 14
| + | |
| − | </span><span style='font-size:11.0pt;font-family:"Cambria Math";color:black'>𝜇</span><span
| + | |
| − | style='font-size:11.0pt;font-family:Arial;color:black'>L water</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'> 2
| + | |
| − | </span><span style='font-size:11.0pt;font-family:"Cambria Math";color:black'>𝜇</span><span
| + | |
| − | style='font-size:11.0pt;font-family:Arial;color:black'>L 10x FD buffer</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'> 2
| + | |
| − | </span><span style='font-size:11.0pt;font-family:"Cambria Math";color:black'>𝜇</span><span
| + | |
| − | style='font-size:11.0pt;font-family:Arial;color:black'>L DNA</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal style='text-indent:.5in'><span style='font-size:11.0pt;
| + | |
| − | font-family:Arial;color:black'>1 </span><span style='font-size:11.0pt;
| + | |
| − | font-family:"Cambria Math";color:black'>𝜇</span><span style='font-size:
| + | |
| − | 11.0pt;font-family:Arial;color:black'>L BglIII</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal style='text-indent:.5in'><u><span style='font-size:11.0pt;
| + | |
| − | font-family:Arial;color:black'>1 </span></u><u><span style='font-size:11.0pt;
| + | |
| − | font-family:"Cambria Math";color:black'>𝜇</span></u><u><span
| + | |
| − | style='font-size:11.0pt;font-family:Arial;color:black'>L </span></u><span
| + | |
| − | style='font-size:11.0pt;font-family:Arial;color:black'>NotI</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal style='text-indent:.5in'><span style='font-size:11.0pt;
| + | |
| − | font-family:Arial;color:black'>20 </span><span style='font-size:11.0pt;
| + | |
| − | font-family:"Cambria Math";color:black'>𝜇</span><span style='font-size:
| + | |
| − | 11.0pt;font-family:Arial;color:black'>L total </span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-family:"Times New Roman"'> </span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'> Sample
| + | |
| − | 2:</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'> 6
| + | |
| − | </span><span style='font-size:11.0pt;font-family:"Cambria Math";color:black'>𝜇</span><span
| + | |
| − | style='font-size:11.0pt;font-family:Arial;color:black'>L water</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'> 2
| + | |
| − | </span><span style='font-size:11.0pt;font-family:"Cambria Math";color:black'>𝜇</span><span
| + | |
| − | style='font-size:11.0pt;font-family:Arial;color:black'>L 10x FD buffer</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'> 10
| + | |
| − | </span><span style='font-size:11.0pt;font-family:"Cambria Math";color:black'>𝜇</span><span
| + | |
| − | style='font-size:11.0pt;font-family:Arial;color:black'>L DNA</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'> 1</span><span
| + | |
| − | style='font-size:11.0pt;font-family:"Cambria Math";color:black'>𝜇</span><span
| + | |
| − | style='font-size:11.0pt;font-family:Arial;color:black'>L BglII</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'> <u>1
| + | |
| − | </u></span><u><span style='font-size:11.0pt;font-family:"Cambria Math";
| + | |
| − | color:black'>𝜇</span></u><u><span style='font-size:11.0pt;font-family:
| + | |
| − | Arial;color:black'>L </span></u><span style='font-size:11.0pt;font-family:Arial;
| + | |
| − | color:black'>NotI</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'> 20
| + | |
| − | </span><span style='font-size:11.0pt;font-family:"Cambria Math";color:black'>𝜇</span><span
| + | |
| − | style='font-size:11.0pt;font-family:Arial;color:black'>L total</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-family:"Times New Roman"'> </span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'> Incubated
| + | |
| − | for 30 minutes at 37 degrees C</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'> Heat
| + | |
| − | inactivated for 5 minutes at 80 degrees C </span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'> Did
| + | |
| − | 2 additional reactions using FD Green in place of FD buffer</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-family:"Times New Roman"'> </span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'>Gel
| + | |
| − | Electrophoresis (using new Lonza 1.2% casette)</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'> Well
| + | |
| − | 1: 5 </span><span style='font-size:11.0pt;font-family:"Cambria Math";
| + | |
| − | color:black'>𝜇</span><span style='font-size:11.0pt;font-family:Arial;
| + | |
| − | color:black'>L Lonza Flash gel DNA Master</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'> Well
| + | |
| − | 2: 4 </span><span style='font-size:11.0pt;font-family:"Cambria Math";
| + | |
| − | color:black'>𝜇</span><span style='font-size:11.0pt;font-family:Arial;
| + | |
| − | color:black'>L Sample 1 (2 </span><span style='font-size:11.0pt;font-family:
| + | |
| − | "Cambria Math";color:black'>𝜇</span><span style='font-size:11.0pt;
| + | |
| − | font-family:Arial;color:black'>L DNA) + 1 </span><span style='font-size:11.0pt;
| + | |
| − | font-family:"Cambria Math";color:black'>𝜇</span><span style='font-size:
| + | |
| − | 11.0pt;font-family:Arial;color:black'>L Lonza Flashgel dye</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'> Well
| + | |
| − | 3: 5 </span><span style='font-size:11.0pt;font-family:"Cambria Math";
| + | |
| − | color:black'>𝜇</span><span style='font-size:11.0pt;font-family:Arial;
| + | |
| − | color:black'>L Sample 1 (2 </span><span style='font-size:11.0pt;font-family:
| + | |
| − | "Cambria Math";color:black'>𝜇</span><span style='font-size:11.0pt;
| + | |
| − | font-family:Arial;color:black'>L DNA) + FD Green dye</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'> Well
| + | |
| − | 4: 4 </span><span style='font-size:11.0pt;font-family:"Cambria Math";
| + | |
| − | color:black'>𝜇</span><span style='font-size:11.0pt;font-family:Arial;
| + | |
| − | color:black'>L Sample 2 (10 </span><span style='font-size:11.0pt;font-family:
| + | |
| − | "Cambria Math";color:black'>𝜇</span><span style='font-size:11.0pt;
| + | |
| − | font-family:Arial;color:black'>L DNA) + 1 </span><span style='font-size:11.0pt;
| + | |
| − | font-family:"Cambria Math";color:black'>𝜇</span><span style='font-size:
| + | |
| − | 11.0pt;font-family:Arial;color:black'>L Lonza Flashgel dye</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'> Well
| + | |
| − | 5: 5 </span><span style='font-size:11.0pt;font-family:"Cambria Math";
| + | |
| − | color:black'>𝜇</span><span style='font-size:11.0pt;font-family:Arial;
| + | |
| − | color:black'>L Sample 2 (10 </span><span style='font-size:11.0pt;font-family:
| + | |
| − | "Cambria Math";color:black'>𝜇</span><span style='font-size:11.0pt;
| + | |
| − | font-family:Arial;color:black'>L DNA) + FD Green dye</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-family:"Times New Roman"'> </span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'> It
| + | |
| − | worked! </span><span style='font-size:11.0pt;font-family:Arial;color:#4A86E8'>(Woot!)</span><span
| + | |
| − | style='font-size:11.0pt;font-family:Arial;color:black'> Well 2 gave the
| + | |
| − | cleanest bands. (Bands were expected at 2046 bp, 842 bp, and 596 bp.)</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'> Made
| + | |
| − | new folder in Google Drive called �Gel Photos�; uploaded photo to folder</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'> Note:
| + | |
| − | Ran gel for 6 minutes 40 seconds. Time was too long because top band
| + | |
| − | disappeared in </span><span style='font-size:11.0pt;font-family:Arial;
| + | |
| − | color:red'>****</span><span style='font-size:11.0pt;font-family:Arial;
| + | |
| − | color:black'> seconds</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-family:"Times New Roman"'> </span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'>8/7/16
| + | |
| − | - Isaac, Charlie</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'>Testing
| + | |
| − | BioBasic Single Step Competency solution protocol for single preparation
| + | |
| − | transformations</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'> Made
| + | |
| − | overnight culture of NEB 5-alpha in 2 mL SOB; incubated at 37 degrees C on
| + | |
| − | shake plate for 13 hours</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'> Inoculated
| + | |
| − | overnight culture into 50 mL of SOB; grew at 37 degrees C on shake plate</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'> At
| + | |
| − | 2 hours, took GD600 using 1:10 dilution; reading = 0.21, </span><span
| + | |
| − | style='font-size:11.0pt;font-family:Arial;color:red'>GD600</span><span
| + | |
| − | style='font-size:11.0pt;font-family:Arial;color:black'> = 2.1</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'> Transformed
| + | |
| − | 5 tubes of 1 mL each using 5 tiers of DNA concentration from test kit</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'> Followed
| + | |
| − | BioBasic SSCS protocol for single prep for each tube</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'> Plated
| + | |
| − | samples onto Chlor plates</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'> At
| + | |
| − | 4 hours, tested absorbance; reading = 0.97, </span><span style='font-size:11.0pt;
| + | |
| − | font-family:Arial;color:red'>OD600</span><span style='font-size:11.0pt;
| + | |
| − | font-family:Arial;color:black'> = 9.7</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'> Repeated
| + | |
| − | competent cell test for 4 hours culture (5 tests)</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-family:"Times New Roman"'> </span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'>8/8/16
| + | |
| − | - Josh</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'>Open
| + | |
| − | lab hours after meeting: double digesting to BBa_K77500 plasmid and the
| + | |
| − | R17_OXTR PCR product and cloning the two with a T4 ligase reaction</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'>Plasmid
| + | |
| − | digestion - added in order</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'> 14
| + | |
| − | </span><span style='font-size:11.0pt;font-family:"Cambria Math";color:black'>𝜇</span><span
| + | |
| − | style='font-size:11.0pt;font-family:Arial;color:black'>L H2O</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'> 2
| + | |
| − | </span><span style='font-size:11.0pt;font-family:"Cambria Math";color:black'>𝜇</span><span
| + | |
| − | style='font-size:11.0pt;font-family:Arial;color:black'>L FD buffer</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'> 2
| + | |
| − | </span><span style='font-size:11.0pt;font-family:"Cambria Math";color:black'>𝜇</span><span
| + | |
| − | style='font-size:11.0pt;font-family:Arial;color:black'>L vector DNA (unknown
| + | |
| − | gene)</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'> 1
| + | |
| − | </span><span style='font-size:11.0pt;font-family:"Cambria Math";color:black'>𝜇</span><span
| + | |
| − | style='font-size:11.0pt;font-family:Arial;color:black'>L BamHI</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'> <u>1
| + | |
| − | </u></span><u><span style='font-size:11.0pt;font-family:"Cambria Math";
| + | |
| − | color:black'>𝜇</span></u><u><span style='font-size:11.0pt;font-family:
| + | |
| − | Arial;color:black'>L </span></u><span style='font-size:11.0pt;font-family:Arial;
| + | |
| − | color:black'>NdeI</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'> 20
| + | |
| − | </span><span style='font-size:11.0pt;font-family:"Cambria Math";color:black'>𝜇</span><span
| + | |
| − | style='font-size:11.0pt;font-family:Arial;color:black'>L total</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-family:"Times New Roman"'> </span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'>PCR
| + | |
| − | product digestion</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'> 14
| + | |
| − | </span><span style='font-size:11.0pt;font-family:"Cambria Math";color:black'>𝜇</span><span
| + | |
| − | style='font-size:11.0pt;font-family:Arial;color:black'>L H2O</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal style='text-indent:.5in'><span style='font-size:11.0pt;
| + | |
| − | font-family:Arial;color:black'>2 </span><span style='font-size:11.0pt;
| + | |
| − | font-family:"Cambria Math";color:black'>𝜇</span><span style='font-size:
| + | |
| − | 11.0pt;font-family:Arial;color:black'>L FD buffer</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'> 12
| + | |
| − | </span><span style='font-size:11.0pt;font-family:"Cambria Math";color:black'>𝜇</span><span
| + | |
| − | style='font-size:11.0pt;font-family:Arial;color:black'>L DNA (@ 10 ng/</span><span
| + | |
| − | style='font-size:11.0pt;font-family:Arial;color:red'>***</span><span
| + | |
| − | style='font-size:11.0pt;font-family:Arial;color:black'>)</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'> 1
| + | |
| − | </span><span style='font-size:11.0pt;font-family:"Cambria Math";color:black'>𝜇</span><span
| + | |
| − | style='font-size:11.0pt;font-family:Arial;color:black'>L BamHI</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'> <u>1
| + | |
| − | </u></span><u><span style='font-size:11.0pt;font-family:"Cambria Math";
| + | |
| − | color:black'>𝜇</span></u><u><span style='font-size:11.0pt;font-family:
| + | |
| − | Arial;color:black'>L </span></u><span style='font-size:11.0pt;font-family:Arial;
| + | |
| − | color:black'>NdeI</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'> 30
| + | |
| − | </span><span style='font-size:11.0pt;font-family:"Cambria Math";color:black'>𝜇</span><span
| + | |
| − | style='font-size:11.0pt;font-family:Arial;color:black'>L total Note:
| + | |
| − | 9.375 </span><span style='font-size:11.0pt;font-family:"Cambria Math";
| + | |
| − | color:black'>𝜇</span><span style='font-size:11.0pt;font-family:Arial;
| + | |
| − | color:black'>L of this product will have 7.5 ng of DNA</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-family:"Times New Roman"'> </span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:red'>Incubated
| + | |
| − | the plasmid digest reaction at room temp for 5 minutes</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:red'>Heat
| + | |
| − | inactivate the plasmid digest for 5 minutes</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-family:"Times New Roman"'> </span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'>T4
| + | |
| − | Ligation</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'> 2
| + | |
| − | </span><span style='font-size:11.0pt;font-family:"Cambria Math";color:black'>𝜇</span><span
| + | |
| − | style='font-size:11.0pt;font-family:Arial;color:black'>L 10 T4 Buffer</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'> 50
| + | |
| − | ng vector DNA</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'> 37.5
| + | |
| − | ng insert DNA (~ 10 </span><span style='font-size:11.0pt;font-family:"Cambria Math";
| + | |
| − | color:black'>𝜇</span><span style='font-size:11.0pt;font-family:Arial;
| + | |
| − | color:black'>L)</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'> 2
| + | |
| − | </span><span style='font-size:11.0pt;font-family:"Cambria Math";color:black'>𝜇</span><span
| + | |
| − | style='font-size:11.0pt;font-family:Arial;color:black'>L H2O</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'> 1
| + | |
| − | </span><span style='font-size:11.0pt;font-family:"Cambria Math";color:black'>𝜇</span><span
| + | |
| − | style='font-size:11.0pt;font-family:Arial;color:black'>L T4 ligase</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-family:"Times New Roman"'> </span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'>8/11/16</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'>Transformed
| + | |
| − | clone product of R1-OXTR and BBa_K775000 with Lucigen competent cells according
| + | |
| − | to Lucigen protocol with 40 </span><span style='font-size:11.0pt;font-family:
| + | |
| − | "Cambria Math";color:black'>𝜇</span><span style='font-size:11.0pt;
| + | |
| − | font-family:Arial;color:black'>L of cells; incubated at 37 degrees C</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-family:"Times New Roman"'> </span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'>Transformed
| + | |
| − | BBa_K660004 iLov with Lucigen competent cells (30 </span><span
| + | |
| − | style='font-size:11.0pt;font-family:"Cambria Math";color:black'>𝜇</span><span
| + | |
| − | style='font-size:11.0pt;font-family:Arial;color:black'>L cells); incubated at
| + | |
| − | 37 degrees C</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-family:"Times New Roman"'> </span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'>8/15/16
| + | |
| − | - Isaac, et.al</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'>OXTR
| + | |
| − | Clones</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'>Performed
| + | |
| − | plasmid miniprep on overnight cultures (following BioBasic EZ-10 miniprep kit
| + | |
| − | protocol)</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'> Used
| + | |
| − | 1.8 mL of overnight cultures</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'> Miniprep
| + | |
| − | samples stored at -20 degrees C</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-family:"Times New Roman"'> </span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'>Restriction
| + | |
| − | digest</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal style='text-indent:.5in'><span style='font-size:11.0pt;
| + | |
| − | font-family:Arial;color:black'>8 </span><span style='font-size:11.0pt;
| + | |
| − | font-family:"Cambria Math";color:black'>𝜇</span><span style='font-size:
| + | |
| − | 11.0pt;font-family:Arial;color:black'>L miniprep DNA</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal style='margin-left:.5in'><span style='font-size:11.0pt;
| + | |
| − | font-family:Arial;color:black'>2 </span><span style='font-size:11.0pt;
| + | |
| − | font-family:"Cambria Math";color:black'>𝜇</span><span style='font-size:
| + | |
| − | 11.0pt;font-family:Arial;color:black'>L FD buffer</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal style='margin-left:.5in'><span style='font-size:11.0pt;
| + | |
| − | font-family:Arial;color:black'>1 </span><span style='font-size:11.0pt;
| + | |
| − | font-family:"Cambria Math";color:black'>𝜇</span><span style='font-size:
| + | |
| − | 11.0pt;font-family:Arial;color:black'>L HindIII</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal style='margin-left:.5in'><span style='font-size:11.0pt;
| + | |
| − | font-family:Arial;color:black'>1 </span><span style='font-size:11.0pt;
| + | |
| − | font-family:"Cambria Math";color:black'>𝜇</span><span style='font-size:
| + | |
| − | 11.0pt;font-family:Arial;color:black'>L XbaI</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal style='margin-left:.5in'><u><span style='font-size:11.0pt;
| + | |
| − | font-family:Arial;color:black'>8 </span></u><u><span style='font-size:11.0pt;
| + | |
| − | font-family:"Cambria Math";color:black'>𝜇</span></u><u><span
| + | |
| − | style='font-size:11.0pt;font-family:Arial;color:black'>L </span></u><span
| + | |
| − | style='font-size:11.0pt;font-family:Arial;color:black'>H2O</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal style='margin-left:.5in'><span style='font-size:11.0pt;
| + | |
| − | font-family:Arial;color:black'>20 </span><span style='font-size:11.0pt;
| + | |
| − | font-family:"Cambria Math";color:black'>𝜇</span><span style='font-size:
| + | |
| − | 11.0pt;font-family:Arial;color:black'>L total</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-family:"Times New Roman"'> </span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal style='margin-left:.5in'><span style='font-size:11.0pt;
| + | |
| − | font-family:Arial;color:black'>Incubated samples at room temp (26 degrees C)
| + | |
| − | for 5 minutes</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal style='margin-left:.5in'><span style='font-size:11.0pt;
| + | |
| − | font-family:Arial;color:black'>Heat inactivated samples at 80 degrees C for 20
| + | |
| − | minutes</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'>Run
| + | |
| − | samples on Lonza gel with 4 </span><span style='font-size:11.0pt;font-family:
| + | |
| − | "Cambria Math";color:black'>𝜇</span><span style='font-size:11.0pt;
| + | |
| − | font-family:Arial;color:black'>L restriction product 1 </span><span
| + | |
| − | style='font-size:11.0pt;font-family:"Cambria Math";color:black'>𝜇</span><span
| + | |
| − | style='font-size:11.0pt;font-family:Arial;color:black'>L Lonza loading dye</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-family:"Times New Roman"'> </span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'>Results:
| + | |
| − | No OXTR inserts </span><span style='font-size:11.0pt;font-family:Arial;
| + | |
| − | color:#4A86E8'>:(</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-family:"Times New Roman"'> </span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'>8/16/16
| + | |
| − | - Kelsie</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'>OXTR
| + | |
| − | Clones</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'>Performed
| + | |
| − | plasmid minipreps (BioBasic EZ10 kit)</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'> Used
| + | |
| − | 1.5 mL of overnight culture to extract DNA</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'> Protocol
| + | |
| − | Deviation: Did not use Visual Lyse (optical agent)</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'> Protocol
| + | |
| − | Deviation: Warmed elution buffer to 37 degrees C before use</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal style='text-indent:.5in'><span style='font-size:11.0pt;
| + | |
| − | font-family:Arial;color:black'>Samples stored in -20 degrees C</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-family:"Times New Roman"'> </span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'>Restriction
| + | |
| − | Digest</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'> 10
| + | |
| − | </span><span style='font-size:11.0pt;font-family:"Cambria Math";color:black'>𝜇</span><span
| + | |
| − | style='font-size:11.0pt;font-family:Arial;color:black'>L miniprep DNA</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'> 2
| + | |
| − | </span><span style='font-size:11.0pt;font-family:"Cambria Math";
| + | |
| − | color:black'>𝜇</span><span style='font-size:11.0pt;font-family:Arial;
| + | |
| − | color:black'>L FD green buffer</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'> 1
| + | |
| − | </span><span style='font-size:11.0pt;font-family:"Cambria Math";
| + | |
| − | color:black'>𝜇</span><span style='font-size:11.0pt;font-family:Arial;
| + | |
| − | color:black'>L HindIII</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'> 1
| + | |
| − | </span><span style='font-size:11.0pt;font-family:"Cambria Math";
| + | |
| − | color:black'>𝜇</span><span style='font-size:11.0pt;font-family:Arial;
| + | |
| − | color:black'>L XbaI</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'> <u>6
| + | |
| − | </u></span><u><span style='font-size:11.0pt;font-family:"Cambria Math";
| + | |
| − | color:black'>𝜇</span></u><u><span style='font-size:11.0pt;font-family:
| + | |
| − | Arial;color:black'>L </span></u><span style='font-size:11.0pt;font-family:Arial;
| + | |
| − | color:black'>H2O</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'> 20
| + | |
| − | </span><span style='font-size:11.0pt;font-family:"Cambria Math";color:black'>𝜇</span><span
| + | |
| − | style='font-size:11.0pt;font-family:Arial;color:black'>L total</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-family:"Times New Roman"'> </span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'> Incubated
| + | |
| − | 10 minutes at 37 degrees C</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'> Run
| + | |
| − | out 8 </span><span style='font-size:11.0pt;font-family:"Cambria Math";
| + | |
| − | color:black'>𝜇</span><span style='font-size:11.0pt;font-family:Arial;
| + | |
| − | color:black'>L on Lonza gel</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-family:"Times New Roman"'> </span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'>Results</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'> No
| + | |
| − | OXTR inserts present</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'> All
| + | |
| − | fragments larger than 2500 bp</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-family:"Times New Roman"'> </span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'>8/16/16
| + | |
| − | continued - Isaac</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'>Made
| + | |
| − | overnight cultures in 3 ml LB of:</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'> BBa_J63009
| + | |
| − | yeast vector with RFP</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'> BBa_J63010
| + | |
| − | yeast vector with RFP</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'> BBa_K592009
| + | |
| − | AmilCP Blue</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-family:"Times New Roman"'> </span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal style='text-indent:.5in'><span style='font-size:11.0pt;
| + | |
| − | font-family:Arial;color:black'>Incubated at 37 degrees C on shaker; start time:
| + | |
| − | 19:00</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-family:"Times New Roman"'> </span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'>8/17/16
| + | |
| − | - Isaac</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'>Performed
| + | |
| − | miniprep on overnight culture (Bio Basic EZ10 kit)</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'> Used
| + | |
| − | 1.8 ml of overnight culture</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'> Overnight
| + | |
| − | culture of BBa_J63009 did not grow; reinoculated.</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'> Protocol
| + | |
| − | Deviation: Did not use Visual Lyse (optical agent)</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'> Protocol
| + | |
| − | Deviation: Warmed solution II to 37 degrees C before use</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'> Stored
| + | |
| − | product at -20 degrees C</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-family:"Times New Roman"'> </span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'>Made
| + | |
| − | SOB (200 ml)</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'> 1
| + | |
| − | g yeast extract</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'> 4
| + | |
| − | g Tryptase</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'> 0.116
| + | |
| − | g NaCl</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'> 0.0372
| + | |
| − | g KCl</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'> 0.48
| + | |
| − | g MgSO4</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-family:"Times New Roman"'> </span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'>8/20/16
| + | |
| − | - Heather</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'>From
| + | |
| − | InterLab kit:</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'> 10
| + | |
| − | </span><span style='font-size:11.0pt;font-family:"Cambria Math";color:black'>𝜇</span><span
| + | |
| − | style='font-size:11.0pt;font-family:Arial;color:black'>L of 100 pg/</span><span
| + | |
| − | style='font-size:11.0pt;font-family:"Cambria Math";color:black'>𝜇</span><span
| + | |
| − | style='font-size:11.0pt;font-family:Arial;color:black'>L DNA per tube (used 5
| + | |
| − | tubes)</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-family:"Times New Roman"'> </span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'>Transformations</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal style='text-indent:.5in'><span style='font-size:11.0pt;
| + | |
| − | font-family:Arial;color:black'>Thawed competent cells - NEB 5-alpha, 200 </span><span
| + | |
| − | style='font-size:11.0pt;font-family:"Cambria Math";color:black'>𝜇</span><span
| + | |
| − | style='font-size:11.0pt;font-family:Arial;color:black'>L (1 tube)</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal style='text-indent:.5in'><span style='font-size:11.0pt;
| + | |
| − | font-family:Arial;color:black'>Diluted DNA - added 1 </span><span
| + | |
| − | style='font-size:11.0pt;font-family:"Cambria Math";color:black'>𝜇</span><span
| + | |
| − | style='font-size:11.0pt;font-family:Arial;color:black'>L of DNA and 9 </span><span
| + | |
| − | style='font-size:11.0pt;font-family:"Cambria Math";color:black'>𝜇</span><span
| + | |
| − | style='font-size:11.0pt;font-family:Arial;color:black'>Lof H20 to each tube (DO
| + | |
| − | NOT dilute next time)</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal style='text-indent:.5in'><span style='font-size:11.0pt;
| + | |
| − | font-family:Arial;color:black'>Added 40 </span><span style='font-size:11.0pt;
| + | |
| − | font-family:"Cambria Math";color:black'>𝜇</span><span style='font-size:
| + | |
| − | 11.0pt;font-family:Arial;color:black'>L of competent cells to each tube</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal style='text-indent:.5in'><span style='font-size:11.0pt;
| + | |
| − | font-family:Arial;color:black'>Incubated on ice for 30 minutes</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal style='text-indent:.5in'><span style='font-size:11.0pt;
| + | |
| − | font-family:Arial;color:black'>Heat shocked at 42 degrees C.</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal style='text-indent:.5in'><span style='font-size:11.0pt;
| + | |
| − | font-family:Arial;color:black'>Added 200 </span><span style='font-size:11.0pt;
| + | |
| − | font-family:"Cambria Math";color:black'>𝜇</span><span style='font-size:
| + | |
| − | 11.0pt;font-family:Arial;color:black'>L of SOC to each tube</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal style='text-indent:.5in'><span style='font-size:11.0pt;
| + | |
| − | font-family:Arial;color:black'>Incubated on shaker for 2 hours at 37 degrees C</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal style='text-indent:.5in'><span style='font-size:11.0pt;
| + | |
| − | font-family:Arial;color:black'>Pipetted 20 </span><span style='font-size:11.0pt;
| + | |
| − | font-family:"Cambria Math";color:black'>𝜇</span><span style='font-size:
| + | |
| − | 11.0pt;font-family:Arial;color:black'>L + 200* </span><span style='font-size:
| + | |
| − | 11.0pt;font-family:"Cambria Math";color:black'>𝜇</span><span
| + | |
| − | style='font-size:11.0pt;font-family:Arial;color:black'>L of each sample onto
| + | |
| − | Chlor plates</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal style='text-indent:.5in'><span style='font-size:11.0pt;
| + | |
| − | font-family:Arial;color:black'>*Only did 100 </span><span style='font-size:
| + | |
| − | 11.0pt;font-family:"Cambria Math";color:black'>𝜇</span><span
| + | |
| − | style='font-size:11.0pt;font-family:Arial;color:black'>L of positive and
| + | |
| − | negative controls because there were only small plates left</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal style='text-indent:.5in'><span style='font-size:11.0pt;
| + | |
| − | font-family:Arial;color:black'>Incubated at 37 degrees C overnight (14-18
| + | |
| − | hours)</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-family:"Times New Roman"'> </span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'>8/21/16
| + | |
| − | - Isaac, Charlie, Max, Heather</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'>Started
| + | |
| − | yeast transformations</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'> 10
| + | |
| − | ml of YPD, inoculated with yeast (BY4742)</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'> Incubated
| + | |
| − | overnight at 30 degrees C</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-family:"Times New Roman"'> </span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'>8/22/16
| + | |
| − | - Kelsie</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'>Recloned
| + | |
| − | OXTR: use digest products</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'> Less
| + | |
| − | Vector (LV)</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'> 1
| + | |
| − | </span><span style='font-size:11.0pt;font-family:"Cambria Math";color:black'>𝜇</span><span
| + | |
| − | style='font-size:11.0pt;font-family:Arial;color:black'>L vector (50 ng approx.)</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'> 9
| + | |
| − | </span><span style='font-size:11.0pt;font-family:"Cambria Math";color:black'>𝜇</span><span
| + | |
| − | style='font-size:11.0pt;font-family:Arial;color:black'>L OXTR digest (37.5 ng
| + | |
| − | approx.)</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'> 2
| + | |
| − | </span><span style='font-size:11.0pt;font-family:"Cambria Math";color:black'>𝜇</span><span
| + | |
| − | style='font-size:11.0pt;font-family:Arial;color:black'>L T4 ligase buffer</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'> 1
| + | |
| − | </span><span style='font-size:11.0pt;font-family:"Cambria Math";color:black'>𝜇</span><span
| + | |
| − | style='font-size:11.0pt;font-family:Arial;color:black'>L T4 ligase</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'> <u>7
| + | |
| − | </u></span><u><span style='font-size:11.0pt;font-family:"Cambria Math";
| + | |
| − | color:black'>𝜇</span></u><u><span style='font-size:11.0pt;font-family:
| + | |
| − | Arial;color:black'>L </span></u><span style='font-size:11.0pt;font-family:Arial;
| + | |
| − | color:black'>H2O</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'> 20
| + | |
| − | </span><span style='font-size:11.0pt;font-family:"Cambria Math";color:black'>𝜇</span><span
| + | |
| − | style='font-size:11.0pt;font-family:Arial;color:black'>L total</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-family:"Times New Roman"'> </span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'> More
| + | |
| − | Vector (MV)</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'> 2
| + | |
| − | </span><span style='font-size:11.0pt;font-family:"Cambria Math";color:black'>𝜇</span><span
| + | |
| − | style='font-size:11.0pt;font-family:Arial;color:black'>L vector (50 ng approx.)</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'> 9
| + | |
| − | </span><span style='font-size:11.0pt;font-family:"Cambria Math";color:black'>𝜇</span><span
| + | |
| − | style='font-size:11.0pt;font-family:Arial;color:black'>L OXTR digest (37.5 ng
| + | |
| − | approx.)</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'> 2
| + | |
| − | </span><span style='font-size:11.0pt;font-family:"Cambria Math";color:black'>𝜇</span><span
| + | |
| − | style='font-size:11.0pt;font-family:Arial;color:black'>L T4 ligase buffer</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'> 1
| + | |
| − | </span><span style='font-size:11.0pt;font-family:"Cambria Math";color:black'>𝜇</span><span
| + | |
| − | style='font-size:11.0pt;font-family:Arial;color:black'>L T4 ligase</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'> <u>6
| + | |
| − | </u></span><u><span style='font-size:11.0pt;font-family:"Cambria Math";
| + | |
| − | color:black'>𝜇</span></u><u><span style='font-size:11.0pt;font-family:
| + | |
| − | Arial;color:black'>L </span></u><span style='font-size:11.0pt;font-family:Arial;
| + | |
| − | color:black'>H2O</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'> 20
| + | |
| − | </span><span style='font-size:11.0pt;font-family:"Cambria Math";color:black'>𝜇</span><span
| + | |
| − | style='font-size:11.0pt;font-family:Arial;color:black'>L total</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-family:"Times New Roman"'> </span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'> Incubated
| + | |
| − | at room temp. for 10 minutes</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'> Heat
| + | |
| − | inactivated at 65 degrees C for 5 minutes</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-family:"Times New Roman"'> </span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'>8/30/16
| + | |
| − | - Isaac</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'>Ran
| + | |
| − | gel of low vector clones (LV) of OXTR and BBa_K775000, digested with HindIII
| + | |
| − | and PstI</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'> </span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'>Results:
| + | |
| − | No clones detected</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-family:"Times New Roman"'> </span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'>Digested
| + | |
| − | minipreps of More Vector (MV) clones of OXTR and BBa_K775000</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'> MV1
| + | |
| − | and MV2</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'> 1
| + | |
| − | </span><span style='font-size:11.0pt;font-family:"Cambria Math";color:black'>𝜇</span><span
| + | |
| − | style='font-size:11.0pt;font-family:Arial;color:black'>L HindIII (the-ODIN)</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'> 1
| + | |
| − | </span><span style='font-size:11.0pt;font-family:"Cambria Math";color:black'>𝜇</span><span
| + | |
| − | style='font-size:11.0pt;font-family:Arial;color:black'>L PstI (NEB)</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'> 5
| + | |
| − | </span><span style='font-size:11.0pt;font-family:"Cambria Math";color:black'>𝜇</span><span
| + | |
| − | style='font-size:11.0pt;font-family:Arial;color:black'>L NEB buffer 3.1</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'> 8
| + | |
| − | </span><span style='font-size:11.0pt;font-family:"Cambria Math";color:black'>𝜇</span><span
| + | |
| − | style='font-size:11.0pt;font-family:Arial;color:black'>L plasmid DNA</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'> <u>35
| + | |
| − | </u></span><u><span style='font-size:11.0pt;font-family:"Cambria Math";
| + | |
| − | color:black'>𝜇</span></u><u><span style='font-size:11.0pt;font-family:
| + | |
| − | Arial;color:black'>L </span></u><span style='font-size:11.0pt;font-family:Arial;
| + | |
| − | color:black'>H2O</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'> 50
| + | |
| − | </span><span style='font-size:11.0pt;font-family:"Cambria Math";color:black'>𝜇</span><span
| + | |
| − | style='font-size:11.0pt;font-family:Arial;color:black'>L total</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-family:"Times New Roman"'> </span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'> MV3
| + | |
| − | and MV6</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'> 1
| + | |
| − | </span><span style='font-size:11.0pt;font-family:"Cambria Math";color:black'>𝜇</span><span
| + | |
| − | style='font-size:11.0pt;font-family:Arial;color:black'>L HindIII (the-ODIN)</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'> 1
| + | |
| − | </span><span style='font-size:11.0pt;font-family:"Cambria Math";color:black'>𝜇</span><span
| + | |
| − | style='font-size:11.0pt;font-family:Arial;color:black'>L SpeI</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'> 5
| + | |
| − | </span><span style='font-size:11.0pt;font-family:"Cambria Math";color:black'>𝜇</span><span
| + | |
| − | style='font-size:11.0pt;font-family:Arial;color:black'>L NEB cutsmart</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'> 8
| + | |
| − | </span><span style='font-size:11.0pt;font-family:"Cambria Math";color:black'>𝜇</span><span
| + | |
| − | style='font-size:11.0pt;font-family:Arial;color:black'>L plasmid DNA</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'> <u>35
| + | |
| − | </u></span><u><span style='font-size:11.0pt;font-family:"Cambria Math";
| + | |
| − | color:black'>𝜇</span></u><u><span style='font-size:11.0pt;font-family:
| + | |
| − | Arial;color:black'>L </span></u><span style='font-size:11.0pt;font-family:Arial;
| + | |
| − | color:black'>H2O</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'> 50
| + | |
| − | </span><span style='font-size:11.0pt;font-family:"Cambria Math";color:black'>𝜇</span><span
| + | |
| − | style='font-size:11.0pt;font-family:Arial;color:black'>L total</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'> Incubated
| + | |
| − | at 37 degrees for 30 minutes</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-family:"Times New Roman"'> </span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'>Ran
| + | |
| − | gel at 160 volts; started at 18:15, ended at 19:00</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'> Well
| + | |
| − | #1: NEB ladder</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'> Well
| + | |
| − | #2: 15 </span><span style='font-size:11.0pt;font-family:"Cambria Math";
| + | |
| − | color:black'>𝜇</span><span style='font-size:11.0pt;font-family:Arial;
| + | |
| − | color:black'>L MV1 with no SDS NEB dye</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'> Well
| + | |
| − | #3: 15 </span><span style='font-size:11.0pt;font-family:"Cambria Math";
| + | |
| − | color:black'>𝜇</span><span style='font-size:11.0pt;font-family:Arial;
| + | |
| − | color:black'>L MV2 with NEB dye</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'> Well
| + | |
| − | #4: MV3 with no SDS</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'> Well
| + | |
| − | #5: MV4 with NEB dye</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'> Results:
| + | |
| − | no clones found</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-family:"Times New Roman"'> </span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'>9/1/16
| + | |
| − | - Charlie, Isaac</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'>Plated:
| + | |
| − | BBa_K1652000</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'> BBa_K1690007</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'> BBa_K1690000</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'> BBa_K1696001?</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'> BBa_K1690008</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'> BBa_K1723000</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'> BBa_K1723005</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'> BBa_K775004</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'> BBa_K775005</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'> BBa_K988002</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'> BBa_K169006</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'> BBa_K753000</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'> Addgene
| + | |
| − | - pRSII415</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'> pRSII405</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'> BBa_J630061</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'> BBa_J63010</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-family:"Times New Roman"'> </span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'>9/4/16
| + | |
| − | - Charlie</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'>Set
| + | |
| − | up cultures of above (see 9/1/16) </span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal style='text-indent:.5in'><span style='font-size:11.0pt;
| + | |
| − | font-family:Arial;color:black'>Used 3 ml LB and 3 </span><span
| + | |
| − | style='font-size:11.0pt;font-family:"Cambria Math";color:black'>𝜇</span><span
| + | |
| − | style='font-size:11.0pt;font-family:Arial;color:black'>L of appropriate
| + | |
| − | antibiotic</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal style='text-indent:.5in'><span style='font-size:11.0pt;
| + | |
| − | font-family:Arial;color:black'>Incubated at 37 degrees C</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'>Set
| + | |
| − | up culture of BY4742</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'> Used
| + | |
| − | 10 ml of TPD</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'> Incubated
| + | |
| − | at 30 degrees C</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'>Note:
| + | |
| − | BBa_K988002 (OXTR) was added after the other colonies. There seems to be
| + | |
| − | delayed activity. The colonies were small so it�s uncertain whether the
| + | |
| − | inoculation was entirely successful; time will tell�</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-family:"Times New Roman"'> </span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'>9/11/16
| + | |
| − | - Charlie</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'>Made
| + | |
| − | colonies of:</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'> BY4742
| + | |
| − | YPD 10 ml</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'> pRSII415
| + | |
| − | 3 ml LB</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'> pRSII405
| + | |
| − | 3 ml LB</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-family:"Times New Roman"'> </span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'>9/12/16
| + | |
| − | - Charlie, Ryan, Kerry</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'>3A
| + | |
| − | Assembly</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'>Protocol
| + | |
| − | Deviation: did NOT use BSA</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'>Made:</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal style='text-indent:.5in'><span style='font-size:11.0pt;
| + | |
| − | font-family:Arial;color:black'>Enzyme MasterMix Backbone (5 rxns)</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'> 0.5
| + | |
| − | </span><span style='font-size:11.0pt;font-family:"Cambria Math";color:black'>𝜇</span><span
| + | |
| − | style='font-size:11.0pt;font-family:Arial;color:black'>L EcoRI - HF</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'> 0.5
| + | |
| − | </span><span style='font-size:11.0pt;font-family:"Cambria Math";color:black'>𝜇</span><span
| + | |
| − | style='font-size:11.0pt;font-family:Arial;color:black'>L PstI</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'> 5
| + | |
| − | </span><span style='font-size:11.0pt;font-family:"Cambria Math";color:black'>𝜇</span><span
| + | |
| − | style='font-size:11.0pt;font-family:Arial;color:black'>L Cutsmart NEB buffer</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'> 18.5
| + | |
| − | </span><span style='font-size:11.0pt;font-family:"Cambria Math";color:black'>𝜇</span><span
| + | |
| − | style='font-size:11.0pt;font-family:Arial;color:black'>L dH2O</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-family:"Times New Roman"'> </span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal style='text-indent:.5in'><span style='font-size:11.0pt;
| + | |
| − | font-family:Arial;color:black'>Enzyme MasterMix Part A (5 rxns)</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'> 0.5
| + | |
| − | </span><span style='font-size:11.0pt;font-family:"Cambria Math";color:black'>𝜇</span><span
| + | |
| − | style='font-size:11.0pt;font-family:Arial;color:black'>L EcoRI - HF</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'> 0.5
| + | |
| − | </span><span style='font-size:11.0pt;font-family:"Cambria Math";color:black'>𝜇</span><span
| + | |
| − | style='font-size:11.0pt;font-family:Arial;color:black'>L SpeI</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'> 18.5
| + | |
| − | </span><span style='font-size:11.0pt;font-family:"Cambria Math";color:black'>𝜇</span><span
| + | |
| − | style='font-size:11.0pt;font-family:Arial;color:black'>L dH2O</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-family:"Times New Roman"'> </span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal style='text-indent:.5in'><span style='font-size:11.0pt;
| + | |
| − | font-family:Arial;color:black'>Enzyme Master Mix Part B (5 rxns)</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'> 0.5
| + | |
| − | </span><span style='font-size:11.0pt;font-family:"Cambria Math";color:black'>𝜇</span><span
| + | |
| − | style='font-size:11.0pt;font-family:Arial;color:black'>L XbaI</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'> 0.5
| + | |
| − | </span><span style='font-size:11.0pt;font-family:"Cambria Math";color:black'>𝜇</span><span
| + | |
| − | style='font-size:11.0pt;font-family:Arial;color:black'>L PStI</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'> 18.5
| + | |
| − | </span><span style='font-size:11.0pt;font-family:"Cambria Math";color:black'>𝜇</span><span
| + | |
| − | style='font-size:11.0pt;font-family:Arial;color:black'>L dH2O</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-family:"Times New Roman"'> </span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'>Digestion
| + | |
| − | </span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'> Backbone</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'> 4
| + | |
| − | </span><span style='font-size:11.0pt;font-family:"Cambria Math";color:black'>𝜇</span><span
| + | |
| − | style='font-size:11.0pt;font-family:Arial;color:black'>L Miniprep plasmid
| + | |
| − | backbone pRSII405</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'> 4
| + | |
| − | </span><span style='font-size:11.0pt;font-family:"Cambria Math";color:black'>𝜇</span><span
| + | |
| − | style='font-size:11.0pt;font-family:Arial;color:black'>L backbone MasterMix</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-family:"Times New Roman"'> </span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'> Part
| + | |
| − | A</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'> 4
| + | |
| − | </span><span style='font-size:11.0pt;font-family:"Cambria Math";color:black'>𝜇</span><span
| + | |
| − | style='font-size:11.0pt;font-family:Arial;color:black'>L Miniprep BBa_K1652000
| + | |
| − | (yeast promoter)</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'> 4
| + | |
| − | </span><span style='font-size:11.0pt;font-family:"Cambria Math";color:black'>𝜇</span><span
| + | |
| − | style='font-size:11.0pt;font-family:Arial;color:black'>L Part A MasterMix</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-family:"Times New Roman"'> </span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'> Part
| + | |
| − | B</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'> 4
| + | |
| − | </span><span style='font-size:11.0pt;font-family:"Cambria Math";color:black'>𝜇</span><span
| + | |
| − | style='font-size:11.0pt;font-family:Arial;color:black'>L Miniprep iLOV
| + | |
| − | (9/11/16)</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'> 4
| + | |
| − | </span><span style='font-size:11.0pt;font-family:"Cambria Math";color:black'>𝜇</span><span
| + | |
| − | style='font-size:11.0pt;font-family:Arial;color:black'>L Part B MasterMix</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-family:"Times New Roman"'> </span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'> Incubated
| + | |
| − | samples at 37 degrees C for 30 minutes</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'> Heat
| + | |
| − | inactivated samples at 80 degrees C for 20 minutes</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-family:"Times New Roman"'> </span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'>Ligation</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'> 2
| + | |
| − | </span><span style='font-size:11.0pt;font-family:"Cambria Math";color:black'>𝜇</span><span
| + | |
| − | style='font-size:11.0pt;font-family:Arial;color:black'>L pRSII405 plasmid
| + | |
| − | backbone digest</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'> 2
| + | |
| − | </span><span style='font-size:11.0pt;font-family:"Cambria Math";color:black'>𝜇</span><span
| + | |
| − | style='font-size:11.0pt;font-family:Arial;color:black'>L part A BBa_K1652000
| + | |
| − | digest</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'> 2
| + | |
| − | </span><span style='font-size:11.0pt;font-family:"Cambria Math";color:black'>𝜇</span><span
| + | |
| − | style='font-size:11.0pt;font-family:Arial;color:black'>L part B iLOV digest</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'> 1
| + | |
| − | </span><span style='font-size:11.0pt;font-family:"Cambria Math";color:black'>𝜇</span><span
| + | |
| − | style='font-size:11.0pt;font-family:Arial;color:black'>L T4 Ligase buffer</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'> 0.5
| + | |
| − | </span><span style='font-size:11.0pt;font-family:"Cambria Math";color:black'>𝜇</span><span
| + | |
| − | style='font-size:11.0pt;font-family:Arial;color:black'>L T4 Ligase</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'> <u>2.5
| + | |
| − | </u></span><u><span style='font-size:11.0pt;font-family:"Cambria Math";
| + | |
| − | color:black'>𝜇</span></u><u><span style='font-size:11.0pt;font-family:
| + | |
| − | Arial;color:black'>L </span></u><span style='font-size:11.0pt;font-family:Arial;
| + | |
| − | color:black'>dH2O</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'> 10
| + | |
| − | </span><span style='font-size:11.0pt;font-family:"Cambria Math";color:black'>𝜇</span><span
| + | |
| − | style='font-size:11.0pt;font-family:Arial;color:black'>L total</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-family:"Times New Roman"'> </span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'> Incubated
| + | |
| − | sample at 16 degrees C for 30 minutes</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'> Heat
| + | |
| − | inactivated at 80 degrees C for 20 minutes</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-family:"Times New Roman"'> </span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'>9/19/16
| + | |
| − | - Charlie, Andrew, Kerry, Kelsie</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'>Made
| + | |
| − | Chlor plates</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'>Made
| + | |
| − | Amp plates </span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-family:"Times New Roman"'> </span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'>OXTR
| + | |
| − | digest</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'> 1
| + | |
| − | </span><span style='font-size:11.0pt;font-family:"Cambria Math";color:black'>𝜇</span><span
| + | |
| − | style='font-size:11.0pt;font-family:Arial;color:black'>L NdeI</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'> 1
| + | |
| − | </span><span style='font-size:11.0pt;font-family:"Cambria Math";color:black'>𝜇</span><span
| + | |
| − | style='font-size:11.0pt;font-family:Arial;color:black'>L BamHI</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'> 2
| + | |
| − | </span><span style='font-size:11.0pt;font-family:"Cambria Math";color:black'>𝜇</span><span
| + | |
| − | style='font-size:11.0pt;font-family:Arial;color:black'>L FD buffer (not green)</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'> 14
| + | |
| − | </span><span style='font-size:11.0pt;font-family:"Cambria Math";color:black'>𝜇</span><span
| + | |
| − | style='font-size:11.0pt;font-family:Arial;color:black'>L H2O</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'> <u>12
| + | |
| − | </u></span><u><span style='font-size:11.0pt;font-family:"Cambria Math";
| + | |
| − | color:black'>𝜇</span></u><u><span style='font-size:11.0pt;font-family:
| + | |
| − | Arial;color:black'>L</span></u><span style='font-size:11.0pt;font-family:Arial;
| + | |
| − | color:black'> OXTR (10 ng/</span><span style='font-size:11.0pt;font-family:
| + | |
| − | "Cambria Math";color:black'>𝜇</span><span style='font-size:11.0pt;
| + | |
| − | font-family:Arial;color:black'>L)</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'> 30
| + | |
| − | </span><span style='font-size:11.0pt;font-family:"Cambria Math";color:black'>𝜇</span><span
| + | |
| − | style='font-size:11.0pt;font-family:Arial;color:black'>L total</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-family:"Times New Roman"'> </span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'> Incubated
| + | |
| − | at 37 degrees C for 30 minutes.</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'> Heat
| + | |
| − | inactivated at 80 degrees C for 5 minutes.</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-family:"Times New Roman"'> </span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'>Made
| + | |
| − | TSS-CaCl2</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'> 8.5
| + | |
| − | mL LB</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'> 1
| + | |
| − | g PEG4000</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'> 25
| + | |
| − | mM CaCl2</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'> Added
| + | |
| − | dH2O to 10 mL</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-family:"Times New Roman"'> </span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'> TSS-MgCl2</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'> 8.5
| + | |
| − | ml LB</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'> 1
| + | |
| − | g PEG4000</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'> 50
| + | |
| − | mM MgCl2</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'> Added
| + | |
| − | dH2O to 10 mL</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-family:"Times New Roman"'> </span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'> Filter
| + | |
| − | sterilized</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-family:"Times New Roman"'> </span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'> pH
| + | |
| − | CaCl2 TSS: 6.3; pH MgCl2 = 6.1</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'> Note:
| + | |
| − | Chung CT 1993 calls for pH=6.3</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-family:"Times New Roman"'> </span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'>9/20/16</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'>CRISPR
| + | |
| − | trial</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'> Added
| + | |
| − | 1 </span><span style='font-size:11.0pt;font-family:"Cambria Math";color:black'>𝜇</span><span
| + | |
| − | style='font-size:11.0pt;font-family:Arial;color:black'>L of RFP BBa_J04450 to
| + | |
| − | each tube</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal style='margin-left:1.0in;text-indent:-.25in;vertical-align:
| + | |
| − | baseline'><span style='font-size:11.0pt;font-family:Arial;color:black'>1.<span
| + | |
| − | style='font:7.0pt "Times New Roman"'> </span></span><span
| + | |
| − | style='font-size:11.0pt;font-family:Arial;color:black'>DHS alpha CaCl2</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal style='margin-left:1.0in;text-indent:-.25in;vertical-align:
| + | |
| − | baseline'><span style='font-size:11.0pt;font-family:Arial;color:black'>2.<span
| + | |
| − | style='font:7.0pt "Times New Roman"'> </span></span><span
| + | |
| − | style='font-size:11.0pt;font-family:Arial;color:black'>DHS alpha MgCl2</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal style='margin-left:1.0in;text-indent:-.25in;vertical-align:
| + | |
| − | baseline'><span style='font-size:11.0pt;font-family:Arial;color:black'>3.<span
| + | |
| − | style='font:7.0pt "Times New Roman"'> </span></span><span
| + | |
| − | style='font-size:11.0pt;font-family:Arial;color:black'>HME63 CaCl2</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal style='margin-left:1.0in;text-indent:-.25in;vertical-align:
| + | |
| − | baseline'><span style='font-size:11.0pt;font-family:Arial;color:black'>4.<span
| + | |
| − | style='font:7.0pt "Times New Roman"'> </span></span><span
| + | |
| − | style='font-size:11.0pt;font-family:Arial;color:black'>HME 63 MgCl2</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'> Incubated
| + | |
| − | in fridge (4 degrees C) for 30 minutes</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'> Heat
| + | |
| − | shocked at 42 degrees C for 30 seconds</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'> Added
| + | |
| − | 600 </span><span style='font-size:11.0pt;font-family:"Cambria Math";color:black'>𝜇</span><span
| + | |
| − | style='font-size:11.0pt;font-family:Arial;color:black'>L to each tube </span><span
| + | |
| − | style='font-size:11.0pt;font-family:Arial;color:red'>(600 </span><span
| + | |
| − | style='font-size:11.0pt;font-family:"Cambria Math";color:red'>𝜇</span><span
| + | |
| − | style='font-size:11.0pt;font-family:Arial;color:red'>L what?)</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'> Incubated
| + | |
| − | at 37 degrees C on shaker plate for 2 hours</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'> Plated
| + | |
| − | on Chlor plates, incubated at 37 degrees C</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-family:"Times New Roman"'> </span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'>9/21/16</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'>Re-ligation
| + | |
| − | of OXTR into BBa_775000</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'> 4
| + | |
| − | </span><span style='font-size:11.0pt;font-family:"Cambria Math";color:black'>𝜇</span><span
| + | |
| − | style='font-size:11.0pt;font-family:Arial;color:black'>L T4 Ligase</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'> 4
| + | |
| − | </span><span style='font-size:11.0pt;font-family:"Cambria Math";color:black'>𝜇</span><span
| + | |
| − | style='font-size:11.0pt;font-family:Arial;color:black'>L T4 Ligase Buffer</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'> 10
| + | |
| − | </span><span style='font-size:11.0pt;font-family:"Cambria Math";color:black'>𝜇</span><span
| + | |
| − | style='font-size:11.0pt;font-family:Arial;color:black'>L OXTR digested</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'> <u>24
| + | |
| − | </u></span><u><span style='font-size:11.0pt;font-family:"Cambria Math";
| + | |
| − | color:black'>𝜇</span></u><u><span style='font-size:11.0pt;font-family:
| + | |
| − | Arial;color:black'>L</span></u><span style='font-size:11.0pt;font-family:Arial;
| + | |
| − | color:black'> BBa_K775000 digested</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'> 42
| + | |
| − | </span><span style='font-size:11.0pt;font-family:"Cambria Math";color:black'>𝜇</span><span
| + | |
| − | style='font-size:11.0pt;font-family:Arial;color:black'>L total</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-family:"Times New Roman"'> </span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'>Transformation</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'> 10
| + | |
| − | </span><span style='font-size:11.0pt;font-family:"Cambria Math";color:black'>𝜇</span><span
| + | |
| − | style='font-size:11.0pt;font-family:Arial;color:black'>L of ligation product
| + | |
| − | added to 50 </span><span style='font-size:11.0pt;font-family:"Cambria Math";
| + | |
| − | color:black'>𝜇</span><span style='font-size:11.0pt;font-family:Arial;
| + | |
| − | color:black'>L of OneShot Top10 cells</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'> Incubated
| + | |
| − | on ice for 20 minutes</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'> Heat
| + | |
| − | shocked at 42 degrees for 30 seconds</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'> Incubated
| + | |
| − | on ice for 2 minutes</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'> Added
| + | |
| − | 500 </span><span style='font-size:11.0pt;font-family:"Cambria Math";color:black'>𝜇</span><span
| + | |
| − | style='font-size:11.0pt;font-family:Arial;color:black'>L SOC to tube; incubated
| + | |
| − | at 37 degrees C on shake plate for 1 hour</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'> Plated
| + | |
| − | 200 </span><span style='font-size:11.0pt;font-family:"Cambria Math";color:black'>𝜇</span><span
| + | |
| − | style='font-size:11.0pt;font-family:Arial;color:black'>L on Chlor plate</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-family:"Times New Roman"'> </span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'>mCherry
| + | |
| − | reporter 3A assembly in pRSII415</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'>Parts:
| + | |
| − | 1: TEF2, promoter, BBa_K165037</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'> 2:
| + | |
| − | Kozak sequence, BBa_K165002</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'> 3.
| + | |
| − | mCherry, reporter/*****, BBa_K165004</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'> 4.
| + | |
| − | ADH1, terminator, BBa_K801012</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-family:"Times New Roman"'> </span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'> Parts
| + | |
| − | 1 and 2 were ligated together following:</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'> Restriction
| + | |
| − | digest of 1 with EcoRI and SpeI, using 4 </span><span style='font-size:11.0pt;
| + | |
| − | font-family:"Cambria Math";color:black'>𝜇</span><span style='font-size:
| + | |
| − | 11.0pt;font-family:Arial;color:black'>L DNA + 4 </span><span style='font-size:
| + | |
| − | 11.0pt;font-family:"Cambria Math";color:black'>𝜇</span><span
| + | |
| − | style='font-size:11.0pt;font-family:Arial;color:black'>L mix</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'> Restriction
| + | |
| − | digest of 2 with XbaI and PstI, using 4 </span><span style='font-size:11.0pt;
| + | |
| − | font-family:"Cambria Math";color:black'>𝜇</span><span style='font-size:
| + | |
| − | 11.0pt;font-family:Arial;color:black'>L DNA + 4 </span><span style='font-size:
| + | |
| − | 11.0pt;font-family:"Cambria Math";color:black'>𝜇</span><span
| + | |
| − | style='font-size:11.0pt;font-family:Arial;color:black'>L mix</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal style='text-indent:.5in'><span style='font-size:11.0pt;
| + | |
| − | font-family:Arial;color:black'>Parts 1 and 2 incubated at 37 degrees C for 30
| + | |
| − | minutes, heat shocked at 80 degrees C for 20 minutes</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal style='text-indent:.5in'><span style='font-size:11.0pt;
| + | |
| − | font-family:Arial;color:black'>Parts 1 and 2 ligated at room temp for 10
| + | |
| − | minutes, heat shocked at 80 degrees C for 20 minutes. No plasmid added</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal style='text-indent:.5in'><span style='font-size:11.0pt;
| + | |
| − | font-family:Arial;color:black'>Parts 3 and 4 were ligated following same
| + | |
| − | procedure</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal style='text-indent:.5in'><span style='font-size:11.0pt;
| + | |
| − | font-family:Arial;color:black'>Restriction digest of 3 with EcoRI and SpeI</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal style='text-indent:.5in'><span style='font-size:11.0pt;
| + | |
| − | font-family:Arial;color:black'>Restriction digest of 4 with XbaI and PstI</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-family:"Times New Roman"'> </span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal style='text-indent:.5in'><span style='font-size:11.0pt;
| + | |
| − | font-family:Arial;color:black'>Double parts _ and _ were ligated together
| + | |
| − | following:</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal style='text-indent:.5in'><span style='font-size:11.0pt;
| + | |
| − | font-family:Arial;color:black'>Restriction digest of _ with EcoRI and SpeI,
| + | |
| − | using 6 </span><span style='font-size:11.0pt;font-family:"Cambria Math";
| + | |
| − | color:black'>𝜇</span><span style='font-size:11.0pt;font-family:Arial;
| + | |
| − | color:black'>L DNA + 4 </span><span style='font-size:11.0pt;font-family:"Cambria Math";
| + | |
| − | color:black'>𝜇</span><span style='font-size:11.0pt;font-family:Arial;
| + | |
| − | color:black'>L mix</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal style='text-indent:.5in'><span style='font-size:11.0pt;
| + | |
| − | font-family:Arial;color:black'>Restriction digest of _ with XbaI and PstI,
| + | |
| − | using 6 </span><span style='font-size:11.0pt;font-family:"Cambria Math";
| + | |
| − | color:black'>𝜇</span><span style='font-size:11.0pt;font-family:Arial;
| + | |
| − | color:black'>L DNA + 4 </span><span style='font-size:11.0pt;font-family:"Cambria Math";
| + | |
| − | color:black'>𝜇</span><span style='font-size:11.0pt;font-family:Arial;
| + | |
| − | color:black'>L mix</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal style='text-indent:.5in'><span style='font-size:11.0pt;
| + | |
| − | font-family:Arial;color:black'>Restriction digest of pRSII415 with EcoRI and
| + | |
| − | PstI, using 4 </span><span style='font-size:11.0pt;font-family:"Cambria Math";
| + | |
| − | color:black'>𝜇</span><span style='font-size:11.0pt;font-family:Arial;
| + | |
| − | color:black'>L DNA + 4 </span><span style='font-size:11.0pt;font-family:"Cambria Math";
| + | |
| − | color:black'>𝜇</span><span style='font-size:11.0pt;font-family:Arial;
| + | |
| − | color:black'>L mix</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal style='text-indent:.5in'><span style='font-size:11.0pt;
| + | |
| − | font-family:Arial;color:black'>All parts were incubated at 37 degrees C for 30
| + | |
| − | minutes, heat shocked at 80 degrees C for 20 minutes</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-family:"Times New Roman"'> </span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal style='text-indent:.5in'><span style='font-size:11.0pt;
| + | |
| − | font-family:Arial;color:red'>Insert picture here</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-family:"Times New Roman"'> </span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'>Digested
| + | |
| − | parts _, _, and pRSII415, ligated, incubated at room temperature for 10
| + | |
| − | minutes, heat shocked at 80 degrees C for 20 minutes</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-family:"Times New Roman"'> </span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'>Added:</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'> 2
| + | |
| − | </span><span style='font-size:11.0pt;font-family:"Cambria Math";color:black'>𝜇</span><span
| + | |
| − | style='font-size:11.0pt;font-family:Arial;color:black'>L plasmid</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'> 3
| + | |
| − | </span><span style='font-size:11.0pt;font-family:"Cambria Math";color:black'>𝜇</span><span
| + | |
| − | style='font-size:11.0pt;font-family:Arial;color:black'>L _</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'> 3
| + | |
| − | </span><span style='font-size:11.0pt;font-family:"Cambria Math";color:black'>𝜇</span><span
| + | |
| − | style='font-size:11.0pt;font-family:Arial;color:black'>L _</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'> 0.5
| + | |
| − | </span><span style='font-size:11.0pt;font-family:"Cambria Math";color:black'>𝜇</span><span
| + | |
| − | style='font-size:11.0pt;font-family:Arial;color:black'>L T4</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'> 1
| + | |
| − | </span><span style='font-size:11.0pt;font-family:"Cambria Math";color:black'>𝜇</span><span
| + | |
| − | style='font-size:11.0pt;font-family:Arial;color:black'>L buffer</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-family:"Times New Roman"'> </span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'>Part
| + | |
| − | _ ligated to digested pSBIC3</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'>Part
| + | |
| − | _ ligated to digested pSBIC3</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'> Incubated
| + | |
| − | at room temperature for 10 minutes, heat shocked at 80 degrees C for 20 minutes</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-family:"Times New Roman"'> </span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'>Transformed
| + | |
| − | ligated products into 5a following NEB protocol</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'> 25
| + | |
| − | </span><span style='font-size:11.0pt;font-family:"Cambria Math";color:black'>𝜇</span><span
| + | |
| − | style='font-size:11.0pt;font-family:Arial;color:black'>L cells</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'> 0.4
| + | |
| − | </span><span style='font-size:11.0pt;font-family:"Cambria Math";color:black'>𝜇</span><span
| + | |
| − | style='font-size:11.0pt;font-family:Arial;color:black'>L B-ME</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'> 1
| + | |
| − | </span><span style='font-size:11.0pt;font-family:"Cambria Math";color:black'>𝜇</span><span
| + | |
| − | style='font-size:11.0pt;font-family:Arial;color:black'>L DNA product</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-family:"Times New Roman"'> </span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'>4/22/16
| + | |
| − | - Isaac Bean</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'>Made
| + | |
| − | overnight cultures of:</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'> BBa_K1616002</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'> BBa_K1403003</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'> BBa_K1403012</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'> BBa_K1403009</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-family:"Times New Roman"'> </span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'>Restreaked
| + | |
| − | BBa_K1403006</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-family:"Times New Roman"'> </span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'>Made
| + | |
| − | 250 mM KCl for making SOB</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-family:"Times New Roman"'> </span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'>Made
| + | |
| − | 250 SOB</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'> 1.25
| + | |
| − | g yeast extract</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'> 5
| + | |
| − | g tryptone</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'> 500
| + | |
| − | </span><span style='font-size:11.0pt;font-family:"Cambria Math";color:black'>𝜇</span><span
| + | |
| − | style='font-size:11.0pt;font-family:Arial;color:black'>L 5 M NaCl</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'> 0.6
| + | |
| − | g MgSO4</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'> 2.5
| + | |
| − | mL KCl (250 mM solution)</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'> </span><span
| + | |
| − | style='font-size:11.0pt;font-family:Arial;color:red'>There�s more but I can�t
| + | |
| − | read it in my photos</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-family:"Times New Roman"'> </span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'>Made
| + | |
| − | 5 M NaCl solution </span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'> 30
| + | |
| − | mL H2O + 8.76 </span><span style='font-size:11.0pt;font-family:Arial;
| + | |
| − | color:red'>g</span><span style='font-size:11.0pt;font-family:Arial;color:black'>
| + | |
| − | NaCL</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-family:"Times New Roman"'> </span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'>Made
| + | |
| − | 250 mM KCl solution</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'> 100
| + | |
| − | mL H2O + 1.86 g KCl </span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-family:"Times New Roman"'> </span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'>Autoclaved
| + | |
| − | SOB, H2O bottles on liquid cycle</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-family:"Times New Roman"'> </span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'>Made
| + | |
| − | 9 Strep/Kan plates</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'> 150
| + | |
| − | mL H2O + 10 mL powder (from The ODIN)</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'> Microwaved
| + | |
| − | for 30 second increments until solution cleared</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-family:"Times New Roman"'> </span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'>Made
| + | |
| − | HME63 competent cells</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'> Streaked
| + | |
| − | HME63 on native plates, grown overnight</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'> Picked
| + | |
| − | single colony, added to 2 mL LB, grown overnight at 37 degrees C</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'> Innoculated
| + | |
| − | 300 </span><span style='font-size:11.0pt;font-family:"Cambria Math";color:black'>𝜇</span><span
| + | |
| − | style='font-size:11.0pt;font-family:Arial;color:black'>L of overnight culture
| + | |
| − | 40 mL LB</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'> Grown
| + | |
| − | at room temperature (25 degrees C) until optical identity = 0.8</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'> 30
| + | |
| − | mL culture taken, centrifuged at 4 degrees C for 10 minutes at 1500 rpm,
| + | |
| − | decanted supernatant</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'> Resuspended
| + | |
| − | cell pellet with 3 mL TSS</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'> 10%
| + | |
| − | PEG4000</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'> 5%
| + | |
| − | DMSO</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'> 25
| + | |
| − | mM CaCl2</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'> 85%
| + | |
| − | LB</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'> Alloquoted
| + | |
| − | 100 </span><span style='font-size:11.0pt;font-family:"Cambria Math";color:black'>𝜇</span><span
| + | |
| − | style='font-size:11.0pt;font-family:Arial;color:black'>L into cryo tubes,
| + | |
| − | stored at 4 degrees for CRISPR workshop</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-family:"Times New Roman"'> </span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'>Transformed
| + | |
| − | HME63</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'> 2
| + | |
| − | transformations using 1 </span><span style='font-size:11.0pt;font-family:"Cambria Math";
| + | |
| − | color:black'>𝜇</span><span style='font-size:11.0pt;font-family:Arial;
| + | |
| − | color:black'>L RFP reporter (BBa_J04450)</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'> 1
| + | |
| − | transformation using </span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'> 10
| + | |
| − | </span><span style='font-size:11.0pt;font-family:"Cambria Math";color:black'>𝜇</span><span
| + | |
| − | style='font-size:11.0pt;font-family:Arial;color:black'>L crRNA</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'> 10
| + | |
| − | </span><span style='font-size:11.0pt;font-family:"Cambria Math";color:black'>𝜇</span><span
| + | |
| − | style='font-size:11.0pt;font-family:Arial;color:black'>L cas9/</span><span
| + | |
| − | style='font-size:11.0pt;font-family:Arial;color:red'>****</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'> 10
| + | |
| − | </span><span style='font-size:11.0pt;font-family:"Cambria Math";color:black'>𝜇</span><span
| + | |
| − | style='font-size:11.0pt;font-family:Arial;color:black'>L template (from The
| + | |
| − | ODIN)</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'>For
| + | |
| − | all transformations:</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'> Added
| + | |
| − | DNA, incubated at 4 degrees C for 30 minutes</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'> Heat
| + | |
| − | shocked at 42 degrees C for 30 seconds, incubated on ice for 10 minutes</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'> Added
| + | |
| − | 600 </span><span style='font-size:11.0pt;font-family:"Cambria Math";color:black'>𝜇</span><span
| + | |
| − | style='font-size:11.0pt;font-family:Arial;color:black'>L LB</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'> Incubated
| + | |
| − | at 37 degrees C on shake plate for 4 hours</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'> Plated
| + | |
| − | on Chlor (RFP), Strep/Kan (CRISPR)</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'> Stored
| + | |
| − | plates in incubator at 37 degrees C</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-family:"Times New Roman"'> </span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'>9/23/16
| + | |
| − | - HMU</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'>Made
| + | |
| − | overnight cultures in 3 mL LB</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'> �6a�
| + | |
| − | - TEF2 + Kozak</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'> �6b�
| + | |
| − | - mCherry + ADHI terminator</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'> Incubated
| + | |
| − | cultures at 37 degrees C on shake plate</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-family:"Times New Roman"'> </span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'>9/25/16
| + | |
| − | - Ryan</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'>Mini-prep</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'> Note
| + | |
| − | - mini preped parts for future use; numbers were used for ease of process to
| + | |
| − | code for part numbers.</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'> 7
| + | |
| − | - BBa_K1403012 �Banana smell�</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'> 8
| + | |
| − | - BBa_K864400 1/17H �Ptuc promoter�</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'> 9
| + | |
| − | - BBa_K1403009 �Flower smell�</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'> 10
| + | |
| − | - BBa_K1616002 VVD YFP Nter</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'> 11
| + | |
| − | - BBa_K1903013 �R</span><span style='font-size:11.0pt;font-family:Arial;
| + | |
| − | color:red'>***</span><span style='font-size:11.0pt;font-family:Arial;
| + | |
| − | color:black'> smell�</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'> 12
| + | |
| − | - BBa_K1403003 �Lemon smell�</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-family:"Times New Roman"'> </span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'>9/26
| + | |
| − | - Isaac</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'>Made
| + | |
| − | 1000x Kanamycin stock</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'> Dissolved
| + | |
| − | 500 mg Kanamycin in 10 mL sterile H2O</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'> 0.2
| + | |
| − | filter sterilized</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'> Alloquoted
| + | |
| − | 1 mL into epi tubes; stored in -20 degree freezer</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-family:"Times New Roman"'> </span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'>Made
| + | |
| − | 3A Assembly Enzyme Master Mix</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'> Backbone
| + | |
| − | Mix</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'> 5
| + | |
| − | </span><span style='font-size:11.0pt;font-family:"Cambria Math";color:black'>𝜇</span><span
| + | |
| − | style='font-size:11.0pt;font-family:Arial;color:black'>L NEB buffer 2.1</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'> 0.5
| + | |
| − | </span><span style='font-size:11.0pt;font-family:"Cambria Math";color:black'>𝜇</span><span
| + | |
| − | style='font-size:11.0pt;font-family:Arial;color:black'>L EcoRI - HF</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'> 0.5
| + | |
| − | </span><span style='font-size:11.0pt;font-family:"Cambria Math";color:black'>𝜇</span><span
| + | |
| − | style='font-size:11.0pt;font-family:Arial;color:black'>L PstI</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'> <u>19
| + | |
| − | </u></span><u><span style='font-size:11.0pt;font-family:"Cambria Math";
| + | |
| − | color:black'>𝜇</span></u><u><span style='font-size:11.0pt;font-family:
| + | |
| − | Arial;color:black'>L</span></u><span style='font-size:11.0pt;font-family:Arial;
| + | |
| − | color:black'> dH2O</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'> 25
| + | |
| − | total</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-family:"Times New Roman"'> </span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'> Upstream
| + | |
| − | Mix</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'> 5
| + | |
| − | </span><span style='font-size:11.0pt;font-family:"Cambria Math";color:black'>𝜇</span><span
| + | |
| − | style='font-size:11.0pt;font-family:Arial;color:black'>L NEB buffer 2.1</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'> 0.5
| + | |
| − | </span><span style='font-size:11.0pt;font-family:"Cambria Math";color:black'>𝜇</span><span
| + | |
| − | style='font-size:11.0pt;font-family:Arial;color:black'>L EcoRI - HF</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'> 0.5
| + | |
| − | </span><span style='font-size:11.0pt;font-family:"Cambria Math";color:black'>𝜇</span><span
| + | |
| − | style='font-size:11.0pt;font-family:Arial;color:black'>L SpeI</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'> <u>19
| + | |
| − | </u></span><u><span style='font-size:11.0pt;font-family:"Cambria Math";
| + | |
| − | color:black'>𝜇</span></u><u><span style='font-size:11.0pt;font-family:
| + | |
| − | Arial;color:black'>L</span></u><span style='font-size:11.0pt;font-family:Arial;
| + | |
| − | color:black'> dH2O</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'> 25
| + | |
| − | total</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-family:"Times New Roman"'> </span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'> Downstream
| + | |
| − | Mix</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'> 5
| + | |
| − | </span><span style='font-size:11.0pt;font-family:"Cambria Math";color:black'>𝜇</span><span
| + | |
| − | style='font-size:11.0pt;font-family:Arial;color:black'>L NEB buffer 2.1</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'> 0.5
| + | |
| − | </span><span style='font-size:11.0pt;font-family:"Cambria Math";color:black'>𝜇</span><span
| + | |
| − | style='font-size:11.0pt;font-family:Arial;color:black'>L XbaI</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'> 0.5
| + | |
| − | </span><span style='font-size:11.0pt;font-family:"Cambria Math";color:black'>𝜇</span><span
| + | |
| − | style='font-size:11.0pt;font-family:Arial;color:black'>L PstI</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'> <u>19
| + | |
| − | </u></span><u><span style='font-size:11.0pt;font-family:"Cambria Math";
| + | |
| − | color:black'>𝜇</span></u><u><span style='font-size:11.0pt;font-family:
| + | |
| − | Arial;color:black'>L</span></u><span style='font-size:11.0pt;font-family:Arial;
| + | |
| − | color:black'> dH2O</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'> 25
| + | |
| − | total</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-family:"Times New Roman"'> </span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'>Assemble
| + | |
| − | together first two and last two parts of yeast reported in to psB1K3</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'> Part
| + | |
| − | A: TEF2 promoter (BBa_K165037)</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'> Part
| + | |
| − | B: Kozak (BBa_K165002)</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'> Part
| + | |
| − | C: mCherry (BBa_K165004)</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'> Part
| + | |
| − | D: ADHI terminator (BBa_K801012)</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'> PK:
| + | |
| − | plasmid backbone Kanamycin (pSB1K3)</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-family:"Times New Roman"'> </span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'>Digested
| + | |
| − | parts and linearized pSB1K3</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'> Part
| + | |
| − | A: 4 </span><span style='font-size:11.0pt;font-family:"Cambria Math";
| + | |
| − | color:black'>𝜇</span><span style='font-size:11.0pt;font-family:Arial;
| + | |
| − | color:black'>L DNA</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'> 4
| + | |
| − | </span><span style='font-size:11.0pt;font-family:"Cambria Math";color:black'>𝜇</span><span
| + | |
| − | style='font-size:11.0pt;font-family:Arial;color:black'>L Upstream Master Mix
| + | |
| − | (MM)</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-family:"Times New Roman"'> </span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'> Part
| + | |
| − | B: 4 </span><span style='font-size:11.0pt;font-family:"Cambria Math";
| + | |
| − | color:black'>𝜇</span><span style='font-size:11.0pt;font-family:Arial;
| + | |
| − | color:black'>L DNA</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'> 4
| + | |
| − | </span><span style='font-size:11.0pt;font-family:"Cambria Math";color:black'>𝜇</span><span
| + | |
| − | style='font-size:11.0pt;font-family:Arial;color:black'>L Downstream MM</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-family:"Times New Roman"'> </span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'> Part
| + | |
| − | C: 4 </span><span style='font-size:11.0pt;font-family:"Cambria Math";
| + | |
| − | color:black'>𝜇</span><span style='font-size:11.0pt;font-family:Arial;
| + | |
| − | color:black'>L DNA</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'> 4
| + | |
| − | </span><span style='font-size:11.0pt;font-family:"Cambria Math";color:black'>𝜇</span><span
| + | |
| − | style='font-size:11.0pt;font-family:Arial;color:black'>L Upstream MM</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-family:"Times New Roman"'> </span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'> Part
| + | |
| − | D: 4 </span><span style='font-size:11.0pt;font-family:"Cambria Math";
| + | |
| − | color:black'>𝜇</span><span style='font-size:11.0pt;font-family:Arial;
| + | |
| − | color:black'>L DNA</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'> 4
| + | |
| − | </span><span style='font-size:11.0pt;font-family:"Cambria Math";color:black'>𝜇</span><span
| + | |
| − | style='font-size:11.0pt;font-family:Arial;color:black'>L Downstream MM</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-family:"Times New Roman"'> </span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'> PK 4
| + | |
| − | </span><span style='font-size:11.0pt;font-family:"Cambria Math";color:black'>𝜇</span><span
| + | |
| − | style='font-size:11.0pt;font-family:Arial;color:black'>L linearized pSB1K3</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'> 4
| + | |
| − | </span><span style='font-size:11.0pt;font-family:"Cambria Math";color:black'>𝜇</span><span
| + | |
| − | style='font-size:11.0pt;font-family:Arial;color:black'>L Backbone MM</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-family:"Times New Roman"'> </span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'> Incubated
| + | |
| − | at 37 degrees C for 30 minutes; heat shocked at 80 degrees C for 20 minutes in
| + | |
| − | PCR</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-family:"Times New Roman"'> </span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'>Ligated
| + | |
| − | A+B+PK</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'> 2
| + | |
| − | </span><span style='font-size:11.0pt;font-family:"Cambria Math";color:black'>𝜇</span><span
| + | |
| − | style='font-size:11.0pt;font-family:Arial;color:black'>L PK</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'> 2
| + | |
| − | </span><span style='font-size:11.0pt;font-family:"Cambria Math";color:black'>𝜇</span><span
| + | |
| − | style='font-size:11.0pt;font-family:Arial;color:black'>L Part A</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'> 2
| + | |
| − | </span><span style='font-size:11.0pt;font-family:"Cambria Math";color:black'>𝜇</span><span
| + | |
| − | style='font-size:11.0pt;font-family:Arial;color:black'>L Part B</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'> 1
| + | |
| − | </span><span style='font-size:11.0pt;font-family:"Cambria Math";color:black'>𝜇</span><span
| + | |
| − | style='font-size:11.0pt;font-family:Arial;color:black'>L T4 DNA ligase buffer</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'> 0.5
| + | |
| − | </span><span style='font-size:11.0pt;font-family:"Cambria Math";color:black'>𝜇</span><span
| + | |
| − | style='font-size:11.0pt;font-family:Arial;color:black'>L T4 DNA ligase </span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'> <u>2.5
| + | |
| − | </u></span><u><span style='font-size:11.0pt;font-family:"Cambria Math";
| + | |
| − | color:black'>𝜇</span></u><u><span style='font-size:11.0pt;font-family:
| + | |
| − | Arial;color:black'>L </span></u><span style='font-size:11.0pt;font-family:Arial;
| + | |
| − | color:black'>H2O</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'> 10
| + | |
| − | </span><span style='font-size:11.0pt;font-family:"Cambria Math";color:black'>𝜇</span><span
| + | |
| − | style='font-size:11.0pt;font-family:Arial;color:black'>L total</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-family:"Times New Roman"'> </span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'>Ligated
| + | |
| − | C+D+PK</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'> 2
| + | |
| − | </span><span style='font-size:11.0pt;font-family:"Cambria Math";color:black'>𝜇</span><span
| + | |
| − | style='font-size:11.0pt;font-family:Arial;color:black'>L PK</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'> 2
| + | |
| − | </span><span style='font-size:11.0pt;font-family:"Cambria Math";color:black'>𝜇</span><span
| + | |
| − | style='font-size:11.0pt;font-family:Arial;color:black'>L Part C</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'> 2
| + | |
| − | </span><span style='font-size:11.0pt;font-family:"Cambria Math";color:black'>𝜇</span><span
| + | |
| − | style='font-size:11.0pt;font-family:Arial;color:black'>L Part D</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'> 1
| + | |
| − | </span><span style='font-size:11.0pt;font-family:"Cambria Math";color:black'>𝜇</span><span
| + | |
| − | style='font-size:11.0pt;font-family:Arial;color:black'>L T4 DNA ligase buffer</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'> 0.5
| + | |
| − | </span><span style='font-size:11.0pt;font-family:"Cambria Math";color:black'>𝜇</span><span
| + | |
| − | style='font-size:11.0pt;font-family:Arial;color:black'>L T4 DNA ligase </span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'> <u>2.5
| + | |
| − | </u></span><u><span style='font-size:11.0pt;font-family:"Cambria Math";
| + | |
| − | color:black'>𝜇</span></u><u><span style='font-size:11.0pt;font-family:
| + | |
| − | Arial;color:black'>L </span></u><span style='font-size:11.0pt;font-family:Arial;
| + | |
| − | color:black'>H2O</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'> 10
| + | |
| − | </span><span style='font-size:11.0pt;font-family:"Cambria Math";color:black'>𝜇</span><span
| + | |
| − | style='font-size:11.0pt;font-family:Arial;color:black'>L total</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'> </span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'> Both
| + | |
| − | ligated samples incubated at 25 degrees C for 10 minutes; heat inactivated at
| + | |
| − | 65 degrees C for 10 minutes</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-family:"Times New Roman"'> </span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'>Made
| + | |
| − | LB agar plates</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'> 10
| + | |
| − | g LB agar (Biobasic pre-mix) dissolved in 250 mL H2O</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'> Made
| + | |
| − | 6 native plates</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'> Made
| + | |
| − | 11 Kan plates</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-family:"Times New Roman"'> </span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'>Streaked
| + | |
| − | </span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal style='text-indent:.5in'><span style='font-size:11.0pt;
| + | |
| − | font-family:Arial;color:black'>BL21 (from </span><span style='font-size:11.0pt;
| + | |
| − | font-family:Arial;color:red'>****</span><span style='font-size:11.0pt;
| + | |
| − | font-family:Arial;color:black'> lab)</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'> NEB
| + | |
| − | 5-a (store bought)</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-family:"Times New Roman"'> </span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'>Plated
| + | |
| − | </span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'> Part
| + | |
| − | A+Part B+PK onto Kan plate</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'> Part
| + | |
| − | C+Part D+PK onto Kan plate</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-family:"Times New Roman"'> </span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'>Miniprepped</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'> BBa_R1062
| + | |
| − | (LuxR promoter)</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'> BBa_B0033
| + | |
| − | (Weaker RBS)</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'> BBa_B0011
| + | |
| − | (Lux terminator)</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'> BBa_I12036
| + | |
| − | (Lambda promoter)</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'> BBa_I12006
| + | |
| − | (Lambda promoter)</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'> BBa_B0031
| + | |
| − | (Weak RBS)</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'> BBa_K525998
| + | |
| − | (T7 promoter with RBS)</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'> BBa_K1499004
| + | |
| − | (Cellulose Binding Site)</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'> BBa_I12220
| + | |
| − | (pLac promoter)</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-family:"Times New Roman"'> </span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'> Prepped
| + | |
| − | according to BioBasic </span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'> </span><span
| + | |
| − | style='font-size:11.0pt;font-family:Arial;color:#4A86E8'>With extended
| + | |
| − | centrifuge step to provide white noise over 1st presidential debate.</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:#4A86E8'> Making
| + | |
| − | Biology Great Again.</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal style='margin-bottom:12.0pt'><span style='font-family:"Times New Roman"'> </span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'>9/27/16
| + | |
| − | - Isaac</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'>Digested
| + | |
| − | Czech yeast reporter (BBa_K1690006), cloned into pRSII405 and pRSII415</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal style='text-indent:.5in'><span style='font-size:11.0pt;
| + | |
| − | font-family:Arial;color:black'>Digested pADHI_GFP (BBa_K1690006) using:</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal style='text-indent:.5in'><span style='font-size:11.0pt;
| + | |
| − | font-family:Arial;color:black'> 4
| + | |
| − | </span><span style='font-size:11.0pt;font-family:"Cambria Math";color:black'>𝜇</span><span
| + | |
| − | style='font-size:11.0pt;font-family:Arial;color:black'>L DNA</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal style='text-indent:.5in'><span style='font-size:11.0pt;
| + | |
| − | font-family:Arial;color:black'> 4
| + | |
| − | </span><span style='font-size:11.0pt;font-family:"Cambria Math";color:black'>𝜇</span><span
| + | |
| − | style='font-size:11.0pt;font-family:Arial;color:black'>L backbone MM (EcoRI +
| + | |
| − | PstI)</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal style='text-indent:.5in'><span style='font-size:11.0pt;
| + | |
| − | font-family:Arial;color:black'>Designated �GFP�</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-family:"Times New Roman"'> </span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal style='text-indent:.5in'><span style='font-size:11.0pt;
| + | |
| − | font-family:Arial;color:black'>Digested pRSII405 using:</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal style='text-indent:.5in'><span style='font-size:11.0pt;
| + | |
| − | font-family:Arial;color:black'> 4
| + | |
| − | </span><span style='font-size:11.0pt;font-family:"Cambria Math";color:black'>𝜇</span><span
| + | |
| − | style='font-size:11.0pt;font-family:Arial;color:black'>L DNA</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal style='text-indent:.5in'><span style='font-size:11.0pt;
| + | |
| − | font-family:Arial;color:black'> 4
| + | |
| − | </span><span style='font-size:11.0pt;font-family:"Cambria Math";color:black'>𝜇</span><span
| + | |
| − | style='font-size:11.0pt;font-family:Arial;color:black'>L backbone MM (EcoRI
| + | |
| − | +PstI)</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal style='text-indent:.5in'><span style='font-size:11.0pt;
| + | |
| − | font-family:Arial;color:black'>Designated �405�</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-family:"Times New Roman"'> </span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal style='text-indent:.5in'><span style='font-size:11.0pt;
| + | |
| − | font-family:Arial;color:black'>Digested pRSII415 using:</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal style='text-indent:.5in'><span style='font-size:11.0pt;
| + | |
| − | font-family:Arial;color:black'> 4
| + | |
| − | </span><span style='font-size:11.0pt;font-family:"Cambria Math";color:black'>𝜇</span><span
| + | |
| − | style='font-size:11.0pt;font-family:Arial;color:black'>L DNA</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal style='text-indent:.5in'><span style='font-size:11.0pt;
| + | |
| − | font-family:Arial;color:black'> 4
| + | |
| − | </span><span style='font-size:11.0pt;font-family:"Cambria Math";color:black'>𝜇</span><span
| + | |
| − | style='font-size:11.0pt;font-family:Arial;color:black'>L backbone MM (EcoRI +
| + | |
| − | PstI)</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal style='text-indent:.5in'><span style='font-size:11.0pt;
| + | |
| − | font-family:Arial;color:black'>Designated �415�</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-family:"Times New Roman"'> </span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal style='text-indent:.5in'><span style='font-size:11.0pt;
| + | |
| − | font-family:Arial;color:black'>Incubated at 37 degrees C for 30 minutes, heat
| + | |
| − | inactivated at 80 degrees C for 20 minutes</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-family:"Times New Roman"'> </span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal style='text-indent:.5in'><span style='font-size:11.0pt;
| + | |
| − | font-family:Arial;color:black'>Ligated �GFP� and �405� using:</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal style='text-indent:.5in'><span style='font-size:11.0pt;
| + | |
| − | font-family:Arial;color:black'> 2
| + | |
| − | </span><span style='font-size:11.0pt;font-family:"Cambria Math";color:black'>𝜇</span><span
| + | |
| − | style='font-size:11.0pt;font-family:Arial;color:black'>L GFP</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal style='text-indent:.5in'><span style='font-size:11.0pt;
| + | |
| − | font-family:Arial;color:black'> 2
| + | |
| − | </span><span style='font-size:11.0pt;font-family:"Cambria Math";color:black'>𝜇</span><span
| + | |
| − | style='font-size:11.0pt;font-family:Arial;color:black'>L 405</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal style='text-indent:.5in'><span style='font-size:11.0pt;
| + | |
| − | font-family:Arial;color:black'> 1
| + | |
| − | </span><span style='font-size:11.0pt;font-family:"Cambria Math";color:black'>𝜇</span><span
| + | |
| − | style='font-size:11.0pt;font-family:Arial;color:black'>L T4 DNA Ligase buffer</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal style='text-indent:.5in'><span style='font-size:11.0pt;
| + | |
| − | font-family:Arial;color:black'> 0.5
| + | |
| − | </span><span style='font-size:11.0pt;font-family:"Cambria Math";color:black'>𝜇</span><span
| + | |
| − | style='font-size:11.0pt;font-family:Arial;color:black'>L T4 DNA Ligase</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal style='text-indent:.5in'><span style='font-size:11.0pt;
| + | |
| − | font-family:Arial;color:black'> <u>4.5
| + | |
| − | </u></span><u><span style='font-size:11.0pt;font-family:"Cambria Math";
| + | |
| − | color:black'>𝜇</span></u><u><span style='font-size:11.0pt;font-family:
| + | |
| − | Arial;color:black'>L </span></u><span style='font-size:11.0pt;font-family:Arial;
| + | |
| − | color:black'>H2O</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal style='text-indent:.5in'><span style='font-size:11.0pt;
| + | |
| − | font-family:Arial;color:black'> 10
| + | |
| − | </span><span style='font-size:11.0pt;font-family:"Cambria Math";color:black'>𝜇</span><span
| + | |
| − | style='font-size:11.0pt;font-family:Arial;color:black'>L total</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-family:"Times New Roman"'> </span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal style='text-indent:.5in'><span style='font-size:11.0pt;
| + | |
| − | font-family:Arial;color:black'>Ligated �GFP� and �415� using:</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal style='text-indent:.5in'><span style='font-size:11.0pt;
| + | |
| − | font-family:Arial;color:black'> 2
| + | |
| − | </span><span style='font-size:11.0pt;font-family:"Cambria Math";color:black'>𝜇</span><span
| + | |
| − | style='font-size:11.0pt;font-family:Arial;color:black'>L GFP</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal style='text-indent:.5in'><span style='font-size:11.0pt;
| + | |
| − | font-family:Arial;color:black'> 2
| + | |
| − | </span><span style='font-size:11.0pt;font-family:"Cambria Math";color:black'>𝜇</span><span
| + | |
| − | style='font-size:11.0pt;font-family:Arial;color:black'>L 415</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal style='text-indent:.5in'><span style='font-size:11.0pt;
| + | |
| − | font-family:Arial;color:black'> 1
| + | |
| − | </span><span style='font-size:11.0pt;font-family:"Cambria Math";color:black'>𝜇</span><span
| + | |
| − | style='font-size:11.0pt;font-family:Arial;color:black'>L T4 DNA Ligase buffer</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal style='text-indent:.5in'><span style='font-size:11.0pt;
| + | |
| − | font-family:Arial;color:black'> 0.5
| + | |
| − | </span><span style='font-size:11.0pt;font-family:"Cambria Math";color:black'>𝜇</span><span
| + | |
| − | style='font-size:11.0pt;font-family:Arial;color:black'>L T4 DNA Ligase</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal style='text-indent:.5in'><span style='font-size:11.0pt;
| + | |
| − | font-family:Arial;color:black'> <u>4.5
| + | |
| − | </u></span><u><span style='font-size:11.0pt;font-family:"Cambria Math";
| + | |
| − | color:black'>𝜇</span></u><u><span style='font-size:11.0pt;font-family:
| + | |
| − | Arial;color:black'>L </span></u><span style='font-size:11.0pt;font-family:Arial;
| + | |
| − | color:black'>H2O</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal style='text-indent:.5in'><span style='font-size:11.0pt;
| + | |
| − | font-family:Arial;color:black'> 10
| + | |
| − | </span><span style='font-size:11.0pt;font-family:"Cambria Math";color:black'>𝜇</span><span
| + | |
| − | style='font-size:11.0pt;font-family:Arial;color:black'>L total</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-family:"Times New Roman"'> </span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal style='text-indent:.5in'><span style='font-size:11.0pt;
| + | |
| − | font-family:Arial;color:black'>Incubated at 25 degrees C for 10 minutes,
| + | |
| − | incubated at 65 degrees C for 10 minutes.</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-family:"Times New Roman"'> </span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'>Transformed
| + | |
| − | GFP + 405 and GFP +415 using:</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'> 30
| + | |
| − | </span><span style='font-size:11.0pt;font-family:"Cambria Math";color:black'>𝜇</span><span
| + | |
| − | style='font-size:11.0pt;font-family:Arial;color:black'>L NEB 5-a per
| + | |
| − | transformation</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-family:"Times New Roman"'> </span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'>Results:
| + | |
| − | No DNA from </span><span style='font-size:11.0pt;font-family:Arial;color:red'>something</span><span
| + | |
| − | style='font-size:11.0pt;font-family:Arial;color:black'> :( Stopped transforming</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-family:"Times New Roman"'> </span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'>Results:
| + | |
| − | Possibly DNA! Continued transformation</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-family:"Times New Roman"'> </span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'> Added
| + | |
| − | 0.8 </span><span style='font-size:11.0pt;font-family:"Cambria Math";color:black'>𝜇</span><span
| + | |
| − | style='font-size:11.0pt;font-family:Arial;color:black'>L BME to 50 </span><span
| + | |
| − | style='font-size:11.0pt;font-family:"Cambria Math";color:black'>𝜇</span><span
| + | |
| − | style='font-size:11.0pt;font-family:Arial;color:black'>L NEB</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'> Stored
| + | |
| − | on ice for 10 minutes</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'> Split
| + | |
| − | cells into 2 30 </span><span style='font-size:11.0pt;font-family:"Cambria Math";
| + | |
| − | color:black'>𝜇</span><span style='font-size:11.0pt;font-family:Arial;
| + | |
| − | color:black'>L tubes; added 2 </span><span style='font-size:11.0pt;font-family:
| + | |
| − | "Cambria Math";color:black'>𝜇</span><span style='font-size:11.0pt;
| + | |
| − | font-family:Arial;color:black'>L DNA</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'> Stored
| + | |
| − | on ice for 1 hour</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'> Heat
| + | |
| − | shocked at 42 degrees C for 30 seconds, returned to ice for 10 minutes</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'> Added
| + | |
| − | 950 </span><span style='font-size:11.0pt;font-family:"Cambria Math";color:black'>𝜇</span><span
| + | |
| − | style='font-size:11.0pt;font-family:Arial;color:black'>L SOC</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'> Incubated
| + | |
| − | at 37 degrees on shake plate for 2 hours</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'> Plated
| + | |
| − | on Amp plates</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-family:"Times New Roman"'> </span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'>9/28/16</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'>Made
| + | |
| − | YPD plates using 500 mL recipe</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'> 5
| + | |
| − | g yeast extract</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'> 10
| + | |
| − | g peptone</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'> 10
| + | |
| − | g agar</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'> H20
| + | |
| − | to 475 mL</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-family:"Times New Roman"'> </span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'> Autoclaved
| + | |
| − | (split between 2 flasks) </span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'> Added
| + | |
| − | 25 mL dextrose (filter sterilized)</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-family:"Times New Roman"'> </span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'> Used
| + | |
| − | half of solution to make native plates</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-family:"Times New Roman"'> </span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal style='text-indent:.5in'><span style='font-size:11.0pt;
| + | |
| − | font-family:Arial;color:black'>Once remaining solution cooled to 55 degrees C,
| + | |
| − | added 1 </span><span style='font-size:11.0pt;font-family:"Cambria Math";
| + | |
| − | color:black'>𝜇</span><span style='font-size:11.0pt;font-family:Arial;
| + | |
| − | color:black'>L Amp/mL to make Amp plates</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-family:"Times New Roman"'> </span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'>Made
| + | |
| − | overnight cultures</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'> Made
| + | |
| − | 3 o/n ABPK - promoter + Kozak</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'> 3
| + | |
| − | o/n CDPK - mCherry + terminator</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'> Plated
| + | |
| − | on Kan</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-family:"Times New Roman"'> </span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal style='text-indent:.5in'><span style='font-size:11.0pt;
| + | |
| − | font-family:Arial;color:black'>Made 3 o/n ABPK - promoter + Kozak</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'> 3
| + | |
| − | o/n CDPK - mCherry + terminator</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'> Plated
| + | |
| − | on Amp</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-family:"Times New Roman"'> </span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'>Made
| + | |
| − | competent cells</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'> Inoculated
| + | |
| − | 5 mL LB with DH5 alpha cells (0.45)</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'> Inoculated
| + | |
| − | 5 mL LB with BL21 cells (0.65)</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'>Incuabted
| + | |
| − | cells at 37 degrees on shake plate overnight</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-family:"Times New Roman"'> </span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'>9/29/16</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'>Overnight
| + | |
| − | cultures</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'> Added
| + | |
| − | 100 </span><span style='font-size:11.0pt;font-family:"Cambria Math";color:black'>𝜇</span><span
| + | |
| − | style='font-size:11.0pt;font-family:Arial;color:black'>L of DH5 alpha overnight
| + | |
| − | cultures to 30 mL of LB (x2)</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'> Added
| + | |
| − | 100 </span><span style='font-size:11.0pt;font-family:"Cambria Math";color:black'>𝜇</span><span
| + | |
| − | style='font-size:11.0pt;font-family:Arial;color:black'>L of BL21 overnight
| + | |
| − | culture to 30 mL of LB (x2)</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'> Both
| + | |
| − | cultures placed on shake plate until OD = 0.4 - 0.6</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-family:"Times New Roman"'> </span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'>Used
| + | |
| − | 1 DH5 alpha + BL21 for TSS and DH5 alpha + BL21 for CaCl</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-family:"Times New Roman"'> </span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'>TSS
| + | |
| − | method</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'> Chilled
| + | |
| − | flasks in ice bath for 20 minutes, swirl.</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'> Collected
| + | |
| − | cells by centrifugation at 1200 for 10 minutes at 4 degrees C (done in 4 15 ml
| + | |
| − | tubes: 2 of DH5 alpha, 2 of BL21)</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'> Poured
| + | |
| − | off supernatant</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'> Resuspended
| + | |
| − | pellet in 2.5 mL of TSS</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'> Aliquoted
| + | |
| − | into 1.5 mL tubes</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'> Centrifuged
| + | |
| − | for 10 minutes at 4 degrees C</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal style='text-indent:.5in'><span style='font-size:11.0pt;
| + | |
| − | font-family:Arial;color:black'>Poured off supernatant</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'> Resuspended
| + | |
| − | pellet in 5 mL TSS solution</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'> Aliquoted
| + | |
| − | into 1.5 mL tubes (made 5 tubes of ~500 </span><span style='font-size:11.0pt;
| + | |
| − | font-family:"Cambria Math";color:black'>𝜇</span><span style='font-size:
| + | |
| − | 11.0pt;font-family:Arial;color:black'>L)</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'> Quick
| + | |
| − | freeze dried on dry ice, stored at -80 degrees C</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-family:"Times New Roman"'> </span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'>CaCl
| + | |
| − | method</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'> Placed
| + | |
| − | cells into cold 15 mL tubes (4 15 mL tubes: 2 of DH5 alpha, 2 of BL21)</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'> Placed
| + | |
| − | tubes on ice for 30 minutes</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'> Centrifuged
| + | |
| − | at 3000 rpm for 5 minutes at 4 degrees C</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'> </span><span
| + | |
| − | style='font-size:11.0pt;font-family:Arial;color:#4A86E8'>Had to get dry ice for
| + | |
| − | centrifuge</span><span style='font-size:11.0pt;font-family:Arial;color:black'> </span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'> Poured
| + | |
| − | off supernatant</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'> Resuspended
| + | |
| − | pellet in 10 mL of ice cold 100 mM CaCl2</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'> Stored
| + | |
| − | on ice for almost 2 hours</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'> Centrifuged
| + | |
| − | at 3000 rpm for 5 minutes at 4 degrees C</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'> Poured
| + | |
| − | off supernatant</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'> Resuspended
| + | |
| − | pellet in 1 mL of ice cold 100 mM CaCl2 + 15% glycerol</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'> Placed
| + | |
| − | on ice overnight</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-family:"Times New Roman"'> </span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'>Yeast
| + | |
| − | transformations</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'>Miniprepped
| + | |
| − | earlier in day:</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'> GFP
| + | |
| − | yeast reporter + pRSII415 #1</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'> GPP
| + | |
| − | yeast reporter + pRSII405 #1</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-family:"Times New Roman"'> </span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'> (Picked
| + | |
| − | up after 20 mL YPD + 1 mL cells BY4742 had been grown for ~6 hours)</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-family:"Times New Roman"'> </span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'> Made
| + | |
| − | 4 tubes of 5 mL</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'> Centrifuged
| + | |
| − | at 3000 rpm for 5 minutes, decanted off supernatant</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal style='text-indent:.5in'><span style='font-size:11.0pt;
| + | |
| − | font-family:Arial;color:black'>Washed cells with H2O</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal style='margin-left:.5in'><span style='font-size:11.0pt;
| + | |
| − | font-family:Arial;color:black'>Centrifuged at 3000 rpm for 5 minutes </span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-family:"Times New Roman"'> </span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'>Made
| + | |
| − | TM Master mix (for 4 transformations)</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'> 240
| + | |
| − | </span><span style='font-size:11.0pt;font-family:"Cambria Math";color:black'>𝜇</span><span
| + | |
| − | style='font-size:11.0pt;font-family:Arial;color:black'>L 50% PEG</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'> 36
| + | |
| − | </span><span style='font-size:11.0pt;font-family:"Cambria Math";color:black'>𝜇</span><span
| + | |
| − | style='font-size:11.0pt;font-family:Arial;color:black'>L IM Lithium Acetate</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'> 25
| + | |
| − | </span><span style='font-size:11.0pt;font-family:"Cambria Math";color:black'>𝜇</span><span
| + | |
| − | style='font-size:11.0pt;font-family:Arial;color:black'>L ssDNA (boiled for 5
| + | |
| − | minutes) </span><span style='font-size:11.0pt;font-family:Arial;color:#4A86E8'>*definitely
| + | |
| − | not done in the InWorks kitchen kettle!</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'> <u>49
| + | |
| − | </u></span><u><span style='font-size:11.0pt;font-family:"Cambria Math";
| + | |
| − | color:black'>𝜇</span></u><u><span style='font-size:11.0pt;font-family:
| + | |
| − | Arial;color:black'>L </span></u><span style='font-size:11.0pt;font-family:Arial;
| + | |
| − | color:black'>H2O</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'> 350
| + | |
| − | </span><span style='font-size:11.0pt;font-family:"Cambria Math";color:black'>𝜇</span><span
| + | |
| − | style='font-size:11.0pt;font-family:Arial;color:black'>L total</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-family:"Times New Roman"'> </span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'> Decanted
| + | |
| − | off supernatant</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'> Resuspended
| + | |
| − | in 200 </span><span style='font-size:11.0pt;font-family:"Cambria Math";
| + | |
| − | color:black'>𝜇</span><span style='font-size:11.0pt;font-family:Arial;
| + | |
| − | color:black'>L of � oops, NOT master mix. Lost 2 tubes</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-family:"Times New Roman"'> </span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'> Resuspended
| + | |
| − | 2 tubes of cells in 1 M lithium acetate</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'> Centrifuged
| + | |
| − | at 13000 rpm for 10 seconds</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'> Decanted
| + | |
| − | off supernatant</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'> Added
| + | |
| − | 80 </span><span style='font-size:11.0pt;font-family:"Cambria Math";color:black'>𝜇</span><span
| + | |
| − | style='font-size:11.0pt;font-family:Arial;color:black'>L of 1 M lithium
| + | |
| − | acetate, vortexed to mix</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'> Made
| + | |
| − | negative controls of 50 </span><span style='font-size:11.0pt;font-family:"Cambria Math";
| + | |
| − | color:black'>𝜇</span><span style='font-size:11.0pt;font-family:Arial;
| + | |
| − | color:black'>L...but then realized we didn�t have enough master mix left after
| + | |
| − | previous �oops�. Discarded controls� :S</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'> Added
| + | |
| − | all remaining master mix to spun down tubes</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'> Added
| + | |
| − | 1 </span><span style='font-size:11.0pt;font-family:"Cambria Math";color:black'>𝜇</span><span
| + | |
| − | style='font-size:11.0pt;font-family:Arial;color:black'>L GFP + pRSII405 #1 to
| + | |
| − | tube #1</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'> Added
| + | |
| − | 1 </span><span style='font-size:11.0pt;font-family:"Cambria Math";color:black'>𝜇</span><span
| + | |
| − | style='font-size:11.0pt;font-family:Arial;color:black'>L GFP + pRSII415 #1 to
| + | |
| − | tube #2</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'> Will
| + | |
| − | do GFP + pRSII405 #2</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'> GFP
| + | |
| − | + pRSII415 #2 on Sunday with other yeast transformations</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'> Incubated
| + | |
| − | both tubes in 30 degrees C water bath for 30 minutes</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'> Incubated
| + | |
| − | tubes in 42 degrees C water bath for 30 minutes</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'> Centrifuged
| + | |
| − | at 13000 rpm for 10 seconds, decanted off supernatant</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'> Added
| + | |
| − | 200 </span><span style='font-size:11.0pt;font-family:"Cambria Math";color:black'>𝜇</span><span
| + | |
| − | style='font-size:11.0pt;font-family:Arial;color:black'>L to tubes, pipetted up
| + | |
| − | and down to mix</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'> Plated
| + | |
| − | 150 </span><span style='font-size:11.0pt;font-family:"Cambria Math";color:black'>𝜇</span><span
| + | |
| − | style='font-size:11.0pt;font-family:Arial;color:black'>L and 50 </span><span
| + | |
| − | style='font-size:11.0pt;font-family:"Cambria Math";color:black'>𝜇</span><span
| + | |
| − | style='font-size:11.0pt;font-family:Arial;color:black'>L on Amp and YPD plates</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-family:"Times New Roman"'> </span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'>3A
| + | |
| − | Assembly of AB (TEF2+Kozak) + CD(mCherry+ADHI) into 405 and 415</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-family:"Times New Roman"'> </span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'>Digested
| + | |
| − | AB (x3):</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'> Designated
| + | |
| − | �AB 1a�</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'> 4
| + | |
| − | </span><span style='font-size:11.0pt;font-family:"Cambria Math";color:black'>𝜇</span><span
| + | |
| − | style='font-size:11.0pt;font-family:Arial;color:black'>L of AB a</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal style='text-indent:.5in'><span style='font-size:11.0pt;
| + | |
| − | font-family:Arial;color:black'> 4
| + | |
| − | </span><span style='font-size:11.0pt;font-family:"Cambria Math";color:black'>𝜇</span><span
| + | |
| − | style='font-size:11.0pt;font-family:Arial;color:black'>L of upstream (U) master
| + | |
| − | mix</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'> </span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'> Designated
| + | |
| − | �AB 1b� </span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'> 4
| + | |
| − | </span><span style='font-size:11.0pt;font-family:"Cambria Math";color:black'>𝜇</span><span
| + | |
| − | style='font-size:11.0pt;font-family:Arial;color:black'>L of AB b</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'> 4
| + | |
| − | </span><span style='font-size:11.0pt;font-family:"Cambria Math";color:black'>𝜇</span><span
| + | |
| − | style='font-size:11.0pt;font-family:Arial;color:black'>L of U MM</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-family:"Times New Roman"'> </span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'> Designated
| + | |
| − | �AB 1c�</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'> 4
| + | |
| − | </span><span style='font-size:11.0pt;font-family:"Cambria Math";color:black'>𝜇</span><span
| + | |
| − | style='font-size:11.0pt;font-family:Arial;color:black'>L of AB c</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'> 4
| + | |
| − | </span><span style='font-size:11.0pt;font-family:"Cambria Math";color:black'>𝜇</span><span
| + | |
| − | style='font-size:11.0pt;font-family:Arial;color:black'>L of U MM</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-family:"Times New Roman"'> </span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'>Digested
| + | |
| − | CD (x3):</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'> Designated
| + | |
| − | �CD 2a�</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'> 4
| + | |
| − | </span><span style='font-size:11.0pt;font-family:"Cambria Math";color:black'>𝜇</span><span
| + | |
| − | style='font-size:11.0pt;font-family:Arial;color:black'>L of CD a</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal style='text-indent:.5in'><span style='font-size:11.0pt;
| + | |
| − | font-family:Arial;color:black'> 4
| + | |
| − | </span><span style='font-size:11.0pt;font-family:"Cambria Math";color:black'>𝜇</span><span
| + | |
| − | style='font-size:11.0pt;font-family:Arial;color:black'>L of downstream (D)
| + | |
| − | master mix</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'> </span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'> Designated
| + | |
| − | �CD 2b� </span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'> 4
| + | |
| − | </span><span style='font-size:11.0pt;font-family:"Cambria Math";color:black'>𝜇</span><span
| + | |
| − | style='font-size:11.0pt;font-family:Arial;color:black'>L of CD b</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'> 4
| + | |
| − | </span><span style='font-size:11.0pt;font-family:"Cambria Math";color:black'>𝜇</span><span
| + | |
| − | style='font-size:11.0pt;font-family:Arial;color:black'>L of D MM</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-family:"Times New Roman"'> </span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'> Designated
| + | |
| − | �CD 2c�</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'> 4
| + | |
| − | </span><span style='font-size:11.0pt;font-family:"Cambria Math";color:black'>𝜇</span><span
| + | |
| − | style='font-size:11.0pt;font-family:Arial;color:black'>L of CD c</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'> 4
| + | |
| − | </span><span style='font-size:11.0pt;font-family:"Cambria Math";color:black'>𝜇</span><span
| + | |
| − | style='font-size:11.0pt;font-family:Arial;color:black'>L of D MM</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-family:"Times New Roman"'> </span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'>Note:
| + | |
| − | 405 and 415 already digested. Used small PCR tubes labelled �405� and �415� in
| + | |
| − | purple sharpie for ligation steps</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'> Incubated
| + | |
| − | samples at 37 degrees C for 30 minutes, heat inactivated at 80 degrees C for 20
| + | |
| − | minutes</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-family:"Times New Roman"'> </span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'>Ligated
| + | |
| − | pSRII405 + AB + CD (x2)</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'> 2
| + | |
| − | </span><span style='font-size:11.0pt;font-family:"Cambria Math";color:black'>𝜇</span><span
| + | |
| − | style='font-size:11.0pt;font-family:Arial;color:black'>L 405</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'> 2
| + | |
| − | </span><span style='font-size:11.0pt;font-family:"Cambria Math";color:black'>𝜇</span><span
| + | |
| − | style='font-size:11.0pt;font-family:Arial;color:black'>L AB 1a</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'> 2
| + | |
| − | </span><span style='font-size:11.0pt;font-family:"Cambria Math";color:black'>𝜇</span><span
| + | |
| − | style='font-size:11.0pt;font-family:Arial;color:black'>L CD 2a </span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'> 1
| + | |
| − | </span><span style='font-size:11.0pt;font-family:"Cambria Math";color:black'>𝜇</span><span
| + | |
| − | style='font-size:11.0pt;font-family:Arial;color:black'>L T4 ligase buffer</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'> 0.5
| + | |
| − | </span><span style='font-size:11.0pt;font-family:"Cambria Math";color:black'>𝜇</span><span
| + | |
| − | style='font-size:11.0pt;font-family:Arial;color:black'>L T4 ligase</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'> <u>2.5
| + | |
| − | </u></span><u><span style='font-size:11.0pt;font-family:"Cambria Math";
| + | |
| − | color:black'>𝜇</span></u><u><span style='font-size:11.0pt;font-family:
| + | |
| − | Arial;color:black'>L </span></u><span style='font-size:11.0pt;font-family:Arial;
| + | |
| − | color:black'>H2O</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'> 10
| + | |
| − | </span><span style='font-size:11.0pt;font-family:"Cambria Math";color:black'>𝜇</span><span
| + | |
| − | style='font-size:11.0pt;font-family:Arial;color:black'> total</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-family:"Times New Roman"'> </span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'> 2
| + | |
| − | </span><span style='font-size:11.0pt;font-family:"Cambria Math";color:black'>𝜇</span><span
| + | |
| − | style='font-size:11.0pt;font-family:Arial;color:black'>L 405</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'> 2
| + | |
| − | </span><span style='font-size:11.0pt;font-family:"Cambria Math";color:black'>𝜇</span><span
| + | |
| − | style='font-size:11.0pt;font-family:Arial;color:black'>L AB 1b</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'> 2
| + | |
| − | </span><span style='font-size:11.0pt;font-family:"Cambria Math";color:black'>𝜇</span><span
| + | |
| − | style='font-size:11.0pt;font-family:Arial;color:black'>L CD 2b </span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'> 1
| + | |
| − | </span><span style='font-size:11.0pt;font-family:"Cambria Math";color:black'>𝜇</span><span
| + | |
| − | style='font-size:11.0pt;font-family:Arial;color:black'>L T4 ligase buffer</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'> 0.5
| + | |
| − | </span><span style='font-size:11.0pt;font-family:"Cambria Math";color:black'>𝜇</span><span
| + | |
| − | style='font-size:11.0pt;font-family:Arial;color:black'>L T4 ligase</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'> <u>2.5
| + | |
| − | </u></span><u><span style='font-size:11.0pt;font-family:"Cambria Math";
| + | |
| − | color:black'>𝜇</span></u><u><span style='font-size:11.0pt;font-family:
| + | |
| − | Arial;color:black'>L </span></u><span style='font-size:11.0pt;font-family:Arial;
| + | |
| − | color:black'>H2O</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'> 10
| + | |
| − | </span><span style='font-size:11.0pt;font-family:"Cambria Math";color:black'>𝜇</span><span
| + | |
| − | style='font-size:11.0pt;font-family:Arial;color:black'> total</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-family:"Times New Roman"'> </span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'>Ligated
| + | |
| − | pSRII415 + AB + CD (x2)</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'> 2
| + | |
| − | </span><span style='font-size:11.0pt;font-family:"Cambria Math";color:black'>𝜇</span><span
| + | |
| − | style='font-size:11.0pt;font-family:Arial;color:black'>L 415</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'> 2
| + | |
| − | </span><span style='font-size:11.0pt;font-family:"Cambria Math";color:black'>𝜇</span><span
| + | |
| − | style='font-size:11.0pt;font-family:Arial;color:black'>L AB 1a</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'> 2
| + | |
| − | </span><span style='font-size:11.0pt;font-family:"Cambria Math";color:black'>𝜇</span><span
| + | |
| − | style='font-size:11.0pt;font-family:Arial;color:black'>L CD 2a </span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'> 1
| + | |
| − | </span><span style='font-size:11.0pt;font-family:"Cambria Math";color:black'>𝜇</span><span
| + | |
| − | style='font-size:11.0pt;font-family:Arial;color:black'>L T4 ligase buffer</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'> 0.5
| + | |
| − | </span><span style='font-size:11.0pt;font-family:"Cambria Math";color:black'>𝜇</span><span
| + | |
| − | style='font-size:11.0pt;font-family:Arial;color:black'>L T4 ligase</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'> <u>2.5
| + | |
| − | </u></span><u><span style='font-size:11.0pt;font-family:"Cambria Math";
| + | |
| − | color:black'>𝜇</span></u><u><span style='font-size:11.0pt;font-family:
| + | |
| − | Arial;color:black'>L </span></u><span style='font-size:11.0pt;font-family:Arial;
| + | |
| − | color:black'>H2O</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'> 10
| + | |
| − | </span><span style='font-size:11.0pt;font-family:"Cambria Math";color:black'>𝜇</span><span
| + | |
| − | style='font-size:11.0pt;font-family:Arial;color:black'>L total</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-family:"Times New Roman"'> </span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'> 2
| + | |
| − | </span><span style='font-size:11.0pt;font-family:"Cambria Math";color:black'>𝜇</span><span
| + | |
| − | style='font-size:11.0pt;font-family:Arial;color:black'>L 415</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'> 2
| + | |
| − | </span><span style='font-size:11.0pt;font-family:"Cambria Math";color:black'>𝜇</span><span
| + | |
| − | style='font-size:11.0pt;font-family:Arial;color:black'>L AB 1b</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'> 2
| + | |
| − | </span><span style='font-size:11.0pt;font-family:"Cambria Math";color:black'>𝜇</span><span
| + | |
| − | style='font-size:11.0pt;font-family:Arial;color:black'>L CD 2b </span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'> 1
| + | |
| − | </span><span style='font-size:11.0pt;font-family:"Cambria Math";color:black'>𝜇</span><span
| + | |
| − | style='font-size:11.0pt;font-family:Arial;color:black'>L T4 ligase buffer</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'> 0.5
| + | |
| − | </span><span style='font-size:11.0pt;font-family:"Cambria Math";color:black'>𝜇</span><span
| + | |
| − | style='font-size:11.0pt;font-family:Arial;color:black'>L T4 ligase</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'> <u>2.5
| + | |
| − | </u></span><u><span style='font-size:11.0pt;font-family:"Cambria Math";
| + | |
| − | color:black'>𝜇</span></u><u><span style='font-size:11.0pt;font-family:
| + | |
| − | Arial;color:black'>L </span></u><span style='font-size:11.0pt;font-family:Arial;
| + | |
| − | color:black'>H2O</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'> 10
| + | |
| − | </span><span style='font-size:11.0pt;font-family:"Cambria Math";color:black'>𝜇</span><span
| + | |
| − | style='font-size:11.0pt;font-family:Arial;color:black'>L total</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-family:"Times New Roman"'> </span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'>Incubated
| + | |
| − | at 2 degrees C for 10 minutes, heat killed at 65 degrees C </span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-family:"Times New Roman"'> </span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'>Transformation
| + | |
| − | (x4)</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'> Thawed
| + | |
| − | 200 </span><span style='font-size:11.0pt;font-family:"Cambria Math";
| + | |
| − | color:black'>𝜇</span><span style='font-size:11.0pt;font-family:Arial;
| + | |
| − | color:black'>L of competent cells (NEB 5 alpha) in 50 mL </span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'> Added
| + | |
| − | 0.8 </span><span style='font-size:11.0pt;font-family:"Cambria Math";color:black'>𝜇</span><span
| + | |
| − | style='font-size:11.0pt;font-family:Arial;color:black'>L BME to cells in new 2
| + | |
| − | mL tubes</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'> Incubated
| + | |
| − | on ice for 10 minutes</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'> Added
| + | |
| − | 1 </span><span style='font-size:11.0pt;font-family:"Cambria Math";color:black'>𝜇</span><span
| + | |
| − | style='font-size:11.0pt;font-family:Arial;color:black'>L DNA to each tube</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'> pRSII405
| + | |
| − | + AB +CD (a)</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'> pRSII405
| + | |
| − | + AB +CD (b)</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'> pRSII415
| + | |
| − | + AB +CD (a)</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'> pRSII415
| + | |
| − | + AB +CD (b)</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'> Incubated
| + | |
| − | on ice for 30 minutes</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'> Heat
| + | |
| − | shocked for 1 minute at 42 degrees C</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'> Added
| + | |
| − | 200 </span><span style='font-size:11.0pt;font-family:"Cambria Math";color:black'>𝜇</span><span
| + | |
| − | style='font-size:11.0pt;font-family:Arial;color:black'>L SOC to each tube</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'> Plated
| + | |
| − | 20 </span><span style='font-size:11.0pt;font-family:"Cambria Math";color:black'>𝜇</span><span
| + | |
| − | style='font-size:11.0pt;font-family:Arial;color:black'>L + 100 </span><span
| + | |
| − | style='font-size:11.0pt;font-family:"Cambria Math";color:black'>𝜇</span><span
| + | |
| − | style='font-size:11.0pt;font-family:Arial;color:black'>L of each transformation
| + | |
| − | on Amp plates</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'> Incubated
| + | |
| − | at 37 degrees C overnight to grow</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-family:"Times New Roman"'> </span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'>10/1/16</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'>Grew
| + | |
| − | overnight cultures of:</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'> AB+CD+405a</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'> AB+CD+405b</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'> AB+CD+415a</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'> AB+CD+415b</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'> GFP
| + | |
| − | in BY4742 +405</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'> GFP
| + | |
| − | in BY4742 +415</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-family:"Times New Roman"'> </span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'>3
| + | |
| − | mL of LB for AB + CD parts with 3 </span><span style='font-size:11.0pt;
| + | |
| − | font-family:"Cambria Math";color:black'>𝜇</span><span style='font-size:
| + | |
| − | 11.0pt;font-family:Arial;color:black'>L of Amp</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'>3
| + | |
| − | mL YPD for GFP parts with 3 </span><span style='font-size:11.0pt;font-family:
| + | |
| − | "Cambria Math";color:black'>𝜇</span><span style='font-size:11.0pt;
| + | |
| − | font-family:Arial;color:black'>L of Amp (</span><span style='font-size:11.0pt;
| + | |
| − | font-family:Arial;color:#4A86E8'>even though this likely won�t work for yeast</span><span
| + | |
| − | style='font-size:11.0pt;font-family:Arial;color:black'>)</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-family:"Times New Roman"'> </span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'>Grew
| + | |
| − | up overnight culture of BY4742 om 10 mL of YPD for yeast transformations
| + | |
| − | tomorrow</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-family:"Times New Roman"'> </span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'>10/2/16</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'>Prepped
| + | |
| − | yeast miniprep kit</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'> Added
| + | |
| − | ethanol to wash solution, added </span><span style='font-size:11.0pt;
| + | |
| − | font-family:Arial;color:red'>snailase(?)</span><span style='font-size:11.0pt;
| + | |
| − | font-family:Arial;color:black'> powder to 0.5 mL of snailase, added isopropanol
| + | |
| − | to DWI buffer</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-family:"Times New Roman"'> </span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'>Started
| + | |
| − | yeast minipreps of GFP+405 and GFP+415</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'> Poured
| + | |
| − | ~1.5 mL of each overnight culture into 2 clean eppendorf tubes</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'> Centrifuged
| + | |
| − | at 10000 rpm for 30 seconds</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'> Added
| + | |
| − | 600 </span><span style='font-size:11.0pt;font-family:"Cambria Math";color:black'>𝜇</span><span
| + | |
| − | style='font-size:11.0pt;font-family:Arial;color:black'>L of snailase reaction
| + | |
| − | buffer</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'> Added
| + | |
| − | 50 </span><span style='font-size:11.0pt;font-family:"Cambria Math";color:black'>𝜇</span><span
| + | |
| − | style='font-size:11.0pt;font-family:Arial;color:black'>L of snailase working
| + | |
| − | stock</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'> Added
| + | |
| − | 1.2 </span><span style='font-size:11.0pt;font-family:"Cambria Math";color:black'>𝜇</span><span
| + | |
| − | style='font-size:11.0pt;font-family:Arial;color:black'>L of mercaptoethanol</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'> Incubated
| + | |
| − | at 37 degrees C for 3 hours</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'> Centrifuged
| + | |
| − | at 5000 rpm for 10 minutes</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'> Added
| + | |
| − | 250 </span><span style='font-size:11.0pt;font-family:"Cambria Math";color:black'>𝜇</span><span
| + | |
| − | style='font-size:11.0pt;font-family:Arial;color:black'>L Buffer P1, vortexed to
| + | |
| − | mix</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'> Added
| + | |
| − | 250 </span><span style='font-size:11.0pt;font-family:"Cambria Math";color:black'>𝜇</span><span
| + | |
| − | style='font-size:11.0pt;font-family:Arial;color:black'>L Buffer P2, incubated
| + | |
| − | at room temperature for 1 minute</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'> Added
| + | |
| − | 350 </span><span style='font-size:11.0pt;font-family:"Cambria Math";color:black'>𝜇</span><span
| + | |
| − | style='font-size:11.0pt;font-family:Arial;color:black'>L Buffer P3, incubated
| + | |
| − | at room temperature for 5 minutes</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'> Centrifuged
| + | |
| − | at 12000 rpm for 10 minutes</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'> Transferred
| + | |
| − | supernatant to spin column, centrifuged at 8000 rpm for 2 minutes</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'> Discarded
| + | |
| − | flow through, then added 500 </span><span style='font-size:11.0pt;font-family:
| + | |
| − | "Cambria Math";color:black'>𝜇</span><span style='font-size:11.0pt;
| + | |
| − | font-family:Arial;color:black'>L buffer DWI, centrifuged at 10000 rpm for 1
| + | |
| − | minute</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'> Discarded
| + | |
| − | flow through, then added 500 </span><span style='font-size:11.0pt;font-family:
| + | |
| − | "Cambria Math";color:black'>𝜇</span><span style='font-size:11.0pt;
| + | |
| − | font-family:Arial;color:black'>L universal wash solution, centrifuged at 10000
| + | |
| − | rpm for 1 minute</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'> Repeated
| + | |
| − | previous step</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'> Discarded
| + | |
| − | flow through, centrifuged for 2 minutes at 10000 rpm to remove residual wash</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'> Transferred
| + | |
| − | spin column to clean eppendorf tube, then added 50 </span><span
| + | |
| − | style='font-size:11.0pt;font-family:"Cambria Math";color:black'>𝜇</span><span
| + | |
| − | style='font-size:11.0pt;font-family:Arial;color:black'>L elution buffer</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'> Incubated
| + | |
| − | at room temperature for 2 minutes, centrifuged at 10000 rpm for 2 minutes</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-family:"Times New Roman"'> </span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'>Miniprepped
| + | |
| − | AB + CD + 405 a+b and AB+ CD + 415 a+b (4 total)</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'> Added
| + | |
| − | 1.5 mL of culture to new eppendorf tubes, centrifuged at 12000 rpm for 2
| + | |
| − | minutes</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'> Added
| + | |
| − | 100 </span><span style='font-size:11.0pt;font-family:"Cambria Math";color:black'>𝜇</span><span
| + | |
| − | style='font-size:11.0pt;font-family:Arial;color:black'>L of Solution 1, let sit
| + | |
| − | for 1 minute </span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'> Added
| + | |
| − | 200 </span><span style='font-size:11.0pt;font-family:"Cambria Math";color:black'>𝜇</span><span
| + | |
| − | style='font-size:11.0pt;font-family:Arial;color:black'>L of Solution 2, let sit
| + | |
| − | for 1 minute</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'> Added
| + | |
| − | 350 </span><span style='font-size:11.0pt;font-family:"Cambria Math";color:black'>𝜇</span><span
| + | |
| − | style='font-size:11.0pt;font-family:Arial;color:black'>L of Solution 3, let sit
| + | |
| − | for 1 minute</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'> Centrifuged
| + | |
| − | at 12000 rpm for 5 minute</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal style='text-indent:.5in'><span style='font-size:11.0pt;
| + | |
| − | font-family:Arial;color:black'>Transferred supernatant to EZ-10 spin columns,
| + | |
| − | centrifuged at 10000 rpm for 2 minutes</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal style='text-indent:.5in'><span style='font-size:11.0pt;
| + | |
| − | font-family:Arial;color:black'>Discarded flow through, added 750 </span><span
| + | |
| − | style='font-size:11.0pt;font-family:"Cambria Math";color:black'>𝜇</span><span
| + | |
| − | style='font-size:11.0pt;font-family:Arial;color:black'>L wash buffer,
| + | |
| − | centrifuged at 10000 rpm for 2 minutes</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal style='text-indent:.5in'><span style='font-size:11.0pt;
| + | |
| − | font-family:Arial;color:black'>Repeated previous step</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal style='text-indent:.5in'><span style='font-size:11.0pt;
| + | |
| − | font-family:Arial;color:black'>Discarded flow through, centrifuged 10000 rpm
| + | |
| − | for 1 minute to remove residual wash</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal style='text-indent:.5in'><span style='font-size:11.0pt;
| + | |
| − | font-family:Arial;color:black'>Transferred columns to new eppendorf tubes</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal style='text-indent:.5in'><span style='font-size:11.0pt;
| + | |
| − | font-family:Arial;color:black'>Added 50 </span><span style='font-size:11.0pt;
| + | |
| − | font-family:"Cambria Math";color:black'>𝜇</span><span style='font-size:
| + | |
| − | 11.0pt;font-family:Arial;color:black'>L of elution buffer, incubated at room
| + | |
| − | temperature for 2 minutes</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal style='text-indent:.5in'><span style='font-size:11.0pt;
| + | |
| − | font-family:Arial;color:black'>Centrifuged at 10000 rpm for 2 minutes</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal style='text-indent:.5in'><span style='font-size:11.0pt;
| + | |
| − | font-family:Arial;color:black'>Readied DNA for diagnostic digests</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal style='text-indent:.5in'><span style='font-size:11.0pt;
| + | |
| − | font-family:Arial;color:black'>Stored purified DNA at -20 degrees C</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-family:"Times New Roman"'> </span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'>Competent
| + | |
| − | cell test on 5 alpha and BL21 with CaCl2 and TSS</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'> Thawed
| + | |
| − | 1 mL aliquots of 5 alpha CaCl2, 5 alpha TSS, BL21 CaCl2, BL21 TSS</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'> Added
| + | |
| − | 50 </span><span style='font-size:11.0pt;font-family:"Cambria Math";color:black'>𝜇</span><span
| + | |
| − | style='font-size:11.0pt;font-family:Arial;color:black'>L of cells to new tubes</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'> Added
| + | |
| − | 1 </span><span style='font-size:11.0pt;font-family:"Cambria Math";color:black'>𝜇</span><span
| + | |
| − | style='font-size:11.0pt;font-family:Arial;color:black'>L of BBa_J04450 (RFP)
| + | |
| − | from comp cell test kit (50 pg/</span><span style='font-size:11.0pt;font-family:
| + | |
| − | "Cambria Math";color:black'>𝜇</span><span style='font-size:11.0pt;
| + | |
| − | font-family:Arial;color:black'>L)</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'> Incubated
| + | |
| − | on ice for 30 minutes</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'> Heat
| + | |
| − | shocked at 42 degrees C for 1 minute</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'> Incubated
| + | |
| − | on ice for 5 minutes</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'> Added
| + | |
| − | 200 </span><span style='font-size:11.0pt;font-family:"Cambria Math";color:black'>𝜇</span><span
| + | |
| − | style='font-size:11.0pt;font-family:Arial;color:black'>L SOC media</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'> Incubated
| + | |
| − | at 37 degrees C for 2 hours</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'> Pipetted
| + | |
| − | 20 </span><span style='font-size:11.0pt;font-family:"Cambria Math";color:black'>𝜇</span><span
| + | |
| − | style='font-size:11.0pt;font-family:Arial;color:black'>L of each onto Chlor
| + | |
| − | plates</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'> Incubated
| + | |
| − | at 37 degrees C overnight.</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-family:"Times New Roman"'> </span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'>10/2/16
| + | |
| − | CJK</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'>Restriction
| + | |
| − | Digest</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'> 10
| + | |
| − | </span><span style='font-size:11.0pt;font-family:"Cambria Math";color:black'>𝜇</span><span
| + | |
| − | style='font-size:11.0pt;font-family:Arial;color:black'>L DNA</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'> 2
| + | |
| − | </span><span style='font-size:11.0pt;font-family:"Cambria Math";color:black'>𝜇</span><span
| + | |
| − | style='font-size:11.0pt;font-family:Arial;color:black'>L FD buffer (not green)</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'> 1
| + | |
| − | </span><span style='font-size:11.0pt;font-family:"Cambria Math";color:black'>𝜇</span><span
| + | |
| − | style='font-size:11.0pt;font-family:Arial;color:black'>L Eco321</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'> <u>7
| + | |
| − | </u></span><u><span style='font-size:11.0pt;font-family:"Cambria Math";
| + | |
| − | color:black'>𝜇</span></u><u><span style='font-size:11.0pt;font-family:
| + | |
| − | Arial;color:black'>L </span></u><span style='font-size:11.0pt;font-family:Arial;
| + | |
| − | color:black'>H2O</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'> 20
| + | |
| − | </span><span style='font-size:11.0pt;font-family:"Cambria Math";color:black'>𝜇</span><span
| + | |
| − | style='font-size:11.0pt;font-family:Arial;color:black'>L total</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-family:"Times New Roman"'> </span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'>Assigned
| + | |
| − | each solution a number:</span></p>
| + | |
| − | | + | |
| − | <ol style='margin-top:0in' start=1 type=1>
| + | |
| − | <li class=MsoNormal style='color:black;vertical-align:baseline'><span
| + | |
| − | style='font-size:11.0pt;font-family:Arial'>AB + CD 405a</span></li>
| + | |
| − | <li class=MsoNormal style='color:black;vertical-align:baseline'><span
| + | |
| − | style='font-size:11.0pt;font-family:Arial'>AB + CD 405b</span></li>
| + | |
| − | <li class=MsoNormal style='color:black;vertical-align:baseline'><span
| + | |
| − | style='font-size:11.0pt;font-family:Arial'>AB + CD 415a</span></li>
| + | |
| − | <li class=MsoNormal style='color:black;vertical-align:baseline'><span
| + | |
| − | style='font-size:11.0pt;font-family:Arial'>AB + CD 415b</span></li>
| + | |
| − | <li class=MsoNormal style='color:black;vertical-align:baseline'><span
| + | |
| − | style='font-size:11.0pt;font-family:Arial'>GFP + 405</span></li>
| + | |
| − | <li class=MsoNormal style='color:black;vertical-align:baseline'><span
| + | |
| − | style='font-size:11.0pt;font-family:Arial'>GFP + 415</span></li>
| + | |
| − | </ol>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-family:"Times New Roman"'> </span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'>Incubated
| + | |
| − | at 37 degrees C for 5 minutes</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-family:"Times New Roman"'> </span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'>Ran
| + | |
| − | gel</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'> 5
| + | |
| − | </span><span style='font-size:11.0pt;font-family:"Cambria Math";color:black'>𝜇</span><span
| + | |
| − | style='font-size:11.0pt;font-family:Arial;color:black'>L DNA</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'> 1
| + | |
| − | </span><span style='font-size:11.0pt;font-family:"Cambria Math";color:black'>𝜇</span><span
| + | |
| − | style='font-size:11.0pt;font-family:Arial;color:black'>L loading dye</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-family:"Times New Roman"'> </span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'>10/3/16
| + | |
| − | CJK, KVP</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'>Repeated
| + | |
| − | procedures from 10/2 with multiple colonies picked from each sample</span></p>
| + | |
| − | | + | |
| − | <ol style='margin-top:0in' start=1 type=1>
| + | |
| − | <li class=MsoNormal style='color:black;vertical-align:baseline'><span
| + | |
| − | style='font-size:11.0pt;font-family:Arial'>AB + CD 405a (1)</span></li>
| + | |
| − | <li class=MsoNormal style='color:black;vertical-align:baseline'><span
| + | |
| − | style='font-size:11.0pt;font-family:Arial'>AB + CD 405b (2)</span></li>
| + | |
| − | <li class=MsoNormal style='color:black;vertical-align:baseline'><span
| + | |
| − | style='font-size:11.0pt;font-family:Arial'>AB + CD 415a (3)</span></li>
| + | |
| − | <li class=MsoNormal style='color:black;vertical-align:baseline'><span
| + | |
| − | style='font-size:11.0pt;font-family:Arial'>AB + CD 415b (4)</span></li>
| + | |
| − | <li class=MsoNormal style='color:black;vertical-align:baseline'><span
| + | |
| − | style='font-size:11.0pt;font-family:Arial'>AB + CD 405a <u>1</u></span></li>
| + | |
| − | <li class=MsoNormal style='color:black;vertical-align:baseline'><span
| + | |
| − | style='font-size:11.0pt;font-family:Arial'>AB + CD 405b <u>2</u></span></li>
| + | |
| − | <li class=MsoNormal style='color:black;vertical-align:baseline'><span
| + | |
| − | style='font-size:11.0pt;font-family:Arial'>AB + CD 415a <u>3</u></span></li>
| + | |
| − | <li class=MsoNormal style='color:black;vertical-align:baseline'><span
| + | |
| − | style='font-size:11.0pt;font-family:Arial'>AB + CD 415b <u>4</u></span></li>
| + | |
| − | </ol>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'>Note:
| + | |
| − | Differentiated groups by underlined or circled numbers</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-family:"Times New Roman"'> </span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'>10/7/16
| + | |
| − | KF</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'>Digest
| + | |
| − | linearized pSB1K3</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'> Enzyme
| + | |
| − | Master Mix</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'> 5
| + | |
| − | </span><span style='font-size:11.0pt;font-family:"Cambria Math";color:black'>𝜇</span><span
| + | |
| − | style='font-size:11.0pt;font-family:Arial;color:black'>L NEB buffer 2.1</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'> 0.5
| + | |
| − | </span><span style='font-size:11.0pt;font-family:"Cambria Math";color:black'>𝜇</span><span
| + | |
| − | style='font-size:11.0pt;font-family:Arial;color:black'>L BSA</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'> 0.5
| + | |
| − | </span><span style='font-size:11.0pt;font-family:"Cambria Math";color:black'>𝜇</span><span
| + | |
| − | style='font-size:11.0pt;font-family:Arial;color:black'>L EcoRI</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'> 0.5
| + | |
| − | </span><span style='font-size:11.0pt;font-family:"Cambria Math";color:black'>𝜇</span><span
| + | |
| − | style='font-size:11.0pt;font-family:Arial;color:black'>L PstI</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'> <u>18.5
| + | |
| − | </u></span><u><span style='font-size:11.0pt;font-family:"Cambria Math";
| + | |
| − | color:black'>𝜇</span></u><u><span style='font-size:11.0pt;font-family:
| + | |
| − | Arial;color:black'>L </span></u><span style='font-size:11.0pt;font-family:Arial;
| + | |
| − | color:black'>H2O</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'> 25
| + | |
| − | </span><span style='font-size:11.0pt;font-family:"Cambria Math";color:black'>𝜇</span><span
| + | |
| − | style='font-size:11.0pt;font-family:Arial;color:black'>L total</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-family:"Times New Roman"'> </span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'> Digest
| + | |
| − | Backbone</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'> Added
| + | |
| − | 4 </span><span style='font-size:11.0pt;font-family:"Cambria Math";color:black'>𝜇</span><span
| + | |
| − | style='font-size:11.0pt;font-family:Arial;color:black'>L linearized backbone
| + | |
| − | (25 ng/</span><span style='font-size:11.0pt;font-family:"Cambria Math";
| + | |
| − | color:black'>𝜇</span><span style='font-size:11.0pt;font-family:Arial;
| + | |
| − | color:black'>L)</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'> Added
| + | |
| − | 4 </span><span style='font-size:11.0pt;font-family:"Cambria Math";color:black'>𝜇</span><span
| + | |
| − | style='font-size:11.0pt;font-family:Arial;color:black'>L enzyme master mix (see
| + | |
| − | above)</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-family:"Times New Roman"'> </span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'> Incubated
| + | |
| − | at 37 degrees C for 30 minutes</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'> Heat
| + | |
| − | inactivated at 80 degrees C for 20 minutes</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'> Antarctic
| + | |
| − | Phosphotase</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'> To
| + | |
| − | digested backbone added:</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'> 2
| + | |
| − | </span><span style='font-size:11.0pt;font-family:"Cambria Math";color:black'>𝜇</span><span
| + | |
| − | style='font-size:11.0pt;font-family:Arial;color:black'>L Ant. Phos. 10x Rxn
| + | |
| − | buffer</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'> 1
| + | |
| − | </span><span style='font-size:11.0pt;font-family:"Cambria Math";color:black'>𝜇</span><span
| + | |
| − | style='font-size:11.0pt;font-family:Arial;color:black'>L Ant. Phos.</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-family:"Times New Roman"'> </span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'> Incubated
| + | |
| − | at 37 degrees C for ~ 15 minutes</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'> Heat
| + | |
| − | inactivated at 70 degrees C for 5 minutes</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'> Extra
| + | |
| − | backbone in Kelsie fridge box: slot A1</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-family:"Times New Roman"'> </span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'>Digests
| + | |
| − | for cloning</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'> OXTR_R17_flag
| + | |
| − | (slot </span><span style='font-size:11.0pt;font-family:Arial;color:red'>J4 Nunc
| + | |
| − | box</span><span style='font-size:11.0pt;font-family:Arial;color:black'>)</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'> 6
| + | |
| − | </span><span style='font-size:11.0pt;font-family:"Cambria Math";color:black'>𝜇</span><span
| + | |
| − | style='font-size:11.0pt;font-family:Arial;color:black'>L H2O</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'> 2
| + | |
| − | </span><span style='font-size:11.0pt;font-family:"Cambria Math";color:black'>𝜇</span><span
| + | |
| − | style='font-size:11.0pt;font-family:Arial;color:black'>L 10x FD buffer</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'> 20
| + | |
| − | </span><span style='font-size:11.0pt;font-family:"Cambria Math";color:black'>𝜇</span><span
| + | |
| − | style='font-size:11.0pt;font-family:Arial;color:black'>L gblock DNA (10 ng/</span><span
| + | |
| − | style='font-size:11.0pt;font-family:"Cambria Math";color:black'>𝜇</span><span
| + | |
| − | style='font-size:11.0pt;font-family:Arial;color:black'>L)</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'> 1
| + | |
| − | </span><span style='font-size:11.0pt;font-family:"Cambria Math";color:black'>𝜇</span><span
| + | |
| − | style='font-size:11.0pt;font-family:Arial;color:black'>L XbaI FD</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'> <u>1
| + | |
| − | </u></span><u><span style='font-size:11.0pt;font-family:"Cambria Math";
| + | |
| − | color:black'>𝜇</span></u><u><span style='font-size:11.0pt;font-family:
| + | |
| − | Arial;color:black'>L </span></u><span style='font-size:11.0pt;font-family:Arial;
| + | |
| − | color:black'>PstI FD</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'> 30
| + | |
| − | </span><span style='font-size:11.0pt;font-family:"Cambria Math";color:black'>𝜇</span><span
| + | |
| − | style='font-size:11.0pt;font-family:Arial;color:black'>L total</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-family:"Times New Roman"'> </span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'> Incubated
| + | |
| − | at 37 degrees C for 30 minutes</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'> Heat
| + | |
| − | inactivated at 65 degrees C for 5 minutes</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'> Extra
| + | |
| − | digest in Kelsie fridge box: slot A2</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'> Note:
| + | |
| − | concentration of OXTR after digest: 6.67ng/</span><span style='font-size:11.0pt;
| + | |
| − | font-family:"Cambria Math";color:black'>𝜇</span><span style='font-size:
| + | |
| − | 11.0pt;font-family:Arial;color:black'>L</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-family:"Times New Roman"'> </span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'> TEF2
| + | |
| − | (slot D6 Nunc box)</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal style='margin-left:.5in;text-indent:.5in'><span
| + | |
| − | style='font-size:11.0pt;font-family:Arial;color:black'>8 </span><span
| + | |
| − | style='font-size:11.0pt;font-family:"Cambria Math";color:black'>𝜇</span><span
| + | |
| − | style='font-size:11.0pt;font-family:Arial;color:black'>L H2O</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal style='margin-left:1.0in'><span style='font-size:11.0pt;
| + | |
| − | font-family:Arial;color:black'>2 </span><span style='font-size:11.0pt;
| + | |
| − | font-family:"Cambria Math";color:black'>𝜇</span><span style='font-size:
| + | |
| − | 11.0pt;font-family:Arial;color:black'>L CutSmart buffer (NEB)</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal style='margin-left:1.0in'><span style='font-size:11.0pt;
| + | |
| − | font-family:Arial;color:black'>8 </span><span style='font-size:11.0pt;
| + | |
| − | font-family:"Cambria Math";color:black'>𝜇</span><span style='font-size:
| + | |
| − | 11.0pt;font-family:Arial;color:black'>L DNA (66.7 ng/</span><span
| + | |
| − | style='font-size:11.0pt;font-family:"Cambria Math";color:black'>𝜇</span><span
| + | |
| − | style='font-size:11.0pt;font-family:Arial;color:black'>L)</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal style='margin-left:1.0in'><span style='font-size:11.0pt;
| + | |
| − | font-family:Arial;color:black'>1 </span><span style='font-size:11.0pt;
| + | |
| − | font-family:"Cambria Math";color:black'>𝜇</span><span style='font-size:
| + | |
| − | 11.0pt;font-family:Arial;color:black'>L EcoRI HF (NEB) </span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal style='margin-left:1.0in'><u><span style='font-size:11.0pt;
| + | |
| − | font-family:Arial;color:black'>1 </span></u><u><span style='font-size:11.0pt;
| + | |
| − | font-family:"Cambria Math";color:black'>𝜇</span></u><u><span
| + | |
| − | style='font-size:11.0pt;font-family:Arial;color:black'>L </span></u><span
| + | |
| − | style='font-size:11.0pt;font-family:Arial;color:black'>SpeI (NEB)</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal style='margin-left:1.0in'><span style='font-size:11.0pt;
| + | |
| − | font-family:Arial;color:black'>20 </span><span style='font-size:11.0pt;
| + | |
| − | font-family:"Cambria Math";color:black'>𝜇</span><span style='font-size:
| + | |
| − | 11.0pt;font-family:Arial;color:black'>L total</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-family:"Times New Roman"'> </span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal style='margin-left:1.0in'><span style='font-size:11.0pt;
| + | |
| − | font-family:Arial;color:black'>Incubated at 37 degrees C for 15 - 30 minutes
| + | |
| − | (30 in the case)</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal style='margin-left:1.0in'><span style='font-size:11.0pt;
| + | |
| − | font-family:Arial;color:black'>Heat inactivated at 65 degrees C for 5 minutes</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal style='margin-left:1.0in'><span style='font-size:11.0pt;
| + | |
| − | font-family:Arial;color:black'>Extra digest in Kelsie fridge box: slot A3</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'> Note:
| + | |
| − | Concentration of TEF2 after digest: 25 ng/</span><span style='font-size:11.0pt;
| + | |
| − | font-family:"Cambria Math";color:black'>𝜇</span><span style='font-size:
| + | |
| − | 11.0pt;font-family:Arial;color:black'>L</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-family:"Times New Roman"'> </span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'> PGK1
| + | |
| − | (slot C6 Nunc box)</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'> 1
| + | |
| − | </span><span style='font-size:11.0pt;font-family:"Cambria Math";color:black'>𝜇</span><span
| + | |
| − | style='font-size:11.0pt;font-family:Arial;color:black'>L H2O</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'> 2
| + | |
| − | </span><span style='font-size:11.0pt;font-family:"Cambria Math";color:black'>𝜇</span><span
| + | |
| − | style='font-size:11.0pt;font-family:Arial;color:black'>L CutSmart buffer (NEB)</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'> 15
| + | |
| − | </span><span style='font-size:11.0pt;font-family:"Cambria Math";color:black'>𝜇</span><span
| + | |
| − | style='font-size:11.0pt;font-family:Arial;color:black'>L DNA (32.4 ng/</span><span
| + | |
| − | style='font-size:11.0pt;font-family:"Cambria Math";color:black'>𝜇</span><span
| + | |
| − | style='font-size:11.0pt;font-family:Arial;color:black'>L)</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'> 1
| + | |
| − | </span><span style='font-size:11.0pt;font-family:"Cambria Math";color:black'>𝜇</span><span
| + | |
| − | style='font-size:11.0pt;font-family:Arial;color:black'>L EcoRI HF (NEB)</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'> <u>1
| + | |
| − | </u></span><u><span style='font-size:11.0pt;font-family:"Cambria Math";
| + | |
| − | color:black'>𝜇</span></u><u><span style='font-size:11.0pt;font-family:
| + | |
| − | Arial;color:black'>L </span></u><span style='font-size:11.0pt;font-family:Arial;
| + | |
| − | color:black'>SpeI (NEB)</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'> 20
| + | |
| − | </span><span style='font-size:11.0pt;font-family:"Cambria Math";color:black'>𝜇</span><span
| + | |
| − | style='font-size:11.0pt;font-family:Arial;color:black'>L total</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-family:"Times New Roman"'> </span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'> Incubated
| + | |
| − | at 37 degrees C for 15 - 30 minutes</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'> Heat
| + | |
| − | inactivated at 65 degrees C for 5 minutes</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'> Extra
| + | |
| − | digest in Kelsie fridge box: slot A4</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'> Note:
| + | |
| − | Concentration of PGK1 after digest: 25 ng/</span><span style='font-size:11.0pt;
| + | |
| − | font-family:"Cambria Math";color:black'>𝜇</span><span style='font-size:
| + | |
| − | 11.0pt;font-family:Arial;color:black'>L</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-family:"Times New Roman"'> </span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'>Ligation</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'> TEF2
| + | |
| − | + OXTR</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'> 2
| + | |
| − | </span><span style='font-size:11.0pt;font-family:"Cambria Math";color:black'>𝜇</span><span
| + | |
| − | style='font-size:11.0pt;font-family:Arial;color:black'>L 10x T4 buffer</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'> 4
| + | |
| − | </span><span style='font-size:11.0pt;font-family:"Cambria Math";color:black'>𝜇</span><span
| + | |
| − | style='font-size:11.0pt;font-family:Arial;color:black'>L pSB1K3 vector DNA (50
| + | |
| − | ng)</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'> 1.5
| + | |
| − | </span><span style='font-size:11.0pt;font-family:"Cambria Math";color:black'>𝜇</span><span
| + | |
| − | style='font-size:11.0pt;font-family:Arial;color:black'>L insert DNA (TEF2 at
| + | |
| − | 37.5 ng)</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'> 5.6
| + | |
| − | </span><span style='font-size:11.0pt;font-family:"Cambria Math";color:black'>𝜇</span><span
| + | |
| − | style='font-size:11.0pt;font-family:Arial;color:black'>L insert DNA (OXTR at
| + | |
| − | 37.5 ng)</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'> 5.9
| + | |
| − | </span><span style='font-size:11.0pt;font-family:"Cambria Math";color:black'>𝜇</span><span
| + | |
| − | style='font-size:11.0pt;font-family:Arial;color:black'>L H2O</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'> <u>1
| + | |
| − | </u></span><u><span style='font-size:11.0pt;font-family:"Cambria Math";
| + | |
| − | color:black'>𝜇</span></u><u><span style='font-size:11.0pt;font-family:
| + | |
| − | Arial;color:black'>L </span></u><span style='font-size:11.0pt;font-family:Arial;
| + | |
| − | color:black'>T4 ligase</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'> 20
| + | |
| − | </span><span style='font-size:11.0pt;font-family:"Cambria Math";color:black'>𝜇</span><span
| + | |
| − | style='font-size:11.0pt;font-family:Arial;color:black'>L total</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal style='margin-bottom:12.0pt'><span style='font-family:"Times New Roman"'> </span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'> PGK1
| + | |
| − | + OXTR</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'> 2
| + | |
| − | </span><span style='font-size:11.0pt;font-family:"Cambria Math";color:black'>𝜇</span><span
| + | |
| − | style='font-size:11.0pt;font-family:Arial;color:black'>L 10x T4 buffer</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'> 4
| + | |
| − | </span><span style='font-size:11.0pt;font-family:"Cambria Math";color:black'>𝜇</span><span
| + | |
| − | style='font-size:11.0pt;font-family:Arial;color:black'>L pSB1K3 vector DNA (50
| + | |
| − | ng)</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'> 1.5
| + | |
| − | </span><span style='font-size:11.0pt;font-family:"Cambria Math";color:black'>𝜇</span><span
| + | |
| − | style='font-size:11.0pt;font-family:Arial;color:black'>L insert DNA (PGK1 at
| + | |
| − | 37.5 ng)</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'> 5.6
| + | |
| − | </span><span style='font-size:11.0pt;font-family:"Cambria Math";color:black'>𝜇</span><span
| + | |
| − | style='font-size:11.0pt;font-family:Arial;color:black'>L insert DNA (OXTR at
| + | |
| − | 37.5 ng)</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'> 5.9
| + | |
| − | </span><span style='font-size:11.0pt;font-family:"Cambria Math";color:black'>𝜇</span><span
| + | |
| − | style='font-size:11.0pt;font-family:Arial;color:black'>L H2O</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'> <u>1
| + | |
| − | </u></span><u><span style='font-size:11.0pt;font-family:"Cambria Math";
| + | |
| − | color:black'>𝜇</span></u><u><span style='font-size:11.0pt;font-family:
| + | |
| − | Arial;color:black'>L </span></u><span style='font-size:11.0pt;font-family:Arial;
| + | |
| − | color:black'>T4 ligase</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'> 20
| + | |
| − | </span><span style='font-size:11.0pt;font-family:"Cambria Math";color:black'>𝜇</span><span
| + | |
| − | style='font-size:11.0pt;font-family:Arial;color:black'>L total</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-family:"Times New Roman"'> </span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'> Incubated
| + | |
| − | at room temperature for 10 minutes</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'> Heat
| + | |
| − | inactivated at 65 degrees C for 5 minutes</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'> Stored
| + | |
| − | in fridge (slots 1 and 2, green rack, 4 degrees C), ready to be transformed</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-family:"Times New Roman"'> </span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'>Digest
| + | |
| − | BBa_K775004 (fast GFP) (slot C3 in Nunc box, -20 degrees C)</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'> BBa_K775004
| + | |
| − | with U enzyme</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'> Added
| + | |
| − | 4 </span><span style='font-size:11.0pt;font-family:"Cambria Math";color:black'>𝜇</span><span
| + | |
| − | style='font-size:11.0pt;font-family:Arial;color:black'>L BBa_K775004 (32.9 ng/</span><span
| + | |
| − | style='font-size:11.0pt;font-family:"Cambria Math";color:black'>𝜇</span><span
| + | |
| − | style='font-size:11.0pt;font-family:Arial;color:black'>L)</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'> Added
| + | |
| − | 4 </span><span style='font-size:11.0pt;font-family:"Cambria Math";color:black'>𝜇</span><span
| + | |
| − | style='font-size:11.0pt;font-family:Arial;color:black'>L U enzyme mix</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-family:"Times New Roman"'> </span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'> Incubated
| + | |
| − | at 37 degrees C for 30 minutes</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'> Heat
| + | |
| − | inactivated at 80 degrees C for 20 minutes</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'> Stored
| + | |
| − | in green rack, slot 3, 4 degrees C</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-family:"Times New Roman"'> </span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal style='text-indent:.5in'><span style='font-size:11.0pt;
| + | |
| − | font-family:Arial;color:black'>BBa_K775004 with D enzyme</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'> Added
| + | |
| − | 4 </span><span style='font-size:11.0pt;font-family:"Cambria Math";color:black'>𝜇</span><span
| + | |
| − | style='font-size:11.0pt;font-family:Arial;color:black'>L BBa_K775004 (32.9 ng/</span><span
| + | |
| − | style='font-size:11.0pt;font-family:"Cambria Math";color:black'>𝜇</span><span
| + | |
| − | style='font-size:11.0pt;font-family:Arial;color:black'>L)</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'> Added
| + | |
| − | 4 </span><span style='font-size:11.0pt;font-family:"Cambria Math";color:black'>𝜇</span><span
| + | |
| − | style='font-size:11.0pt;font-family:Arial;color:black'>L D enzyme mix</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-family:"Times New Roman"'> </span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'> Incubated
| + | |
| − | at 37 degrees C for 30 minutes</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'> Heat
| + | |
| − | inactivated at 80 degrees C for 20 minutes</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'> Stored
| + | |
| − | in green rack, slot 4, 4 degrees C</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-family:"Times New Roman"'> </span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'>Making
| + | |
| − | M9 Minimal media</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'> 1x
| + | |
| − | M9 salts</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'> 2
| + | |
| − | mM MgSO4</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal style='text-indent:.5in'><span style='font-size:11.0pt;
| + | |
| − | font-family:Arial;color:black'> 0.1
| + | |
| − | mM CaCl2</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'> 0.4%
| + | |
| − | carbon source (glycerol, glucose)</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal style='text-indent:.5in'><span style='font-size:11.0pt;
| + | |
| − | font-family:Arial;color:black'> In
| + | |
| − | sterile H2O</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-family:"Times New Roman"'> </span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal style='text-indent:.5in'><span style='font-size:11.0pt;
| + | |
| − | font-family:Arial;color:black'>Made 0.5 L of M9 Salts (5x)</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal style='text-indent:.5in'><span style='font-size:11.0pt;
| + | |
| − | font-family:Arial;color:black'> 32
| + | |
| − | g sodium phosphate dibasic, hepta hydrate</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal style='text-indent:.5in'><span style='font-size:11.0pt;
| + | |
| − | font-family:Arial;color:black'> 75
| + | |
| − | g potassium phosphate</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal style='text-indent:.5in'><span style='font-size:11.0pt;
| + | |
| − | font-family:Arial;color:black'> 1.25
| + | |
| − | g sodium chloride</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal style='text-indent:.5in'><span style='font-size:11.0pt;
| + | |
| − | font-family:Arial;color:black'> 2.5
| + | |
| − | g ammonium chloride</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-family:"Times New Roman"'> </span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal style='text-indent:.5in'><span style='font-size:11.0pt;
| + | |
| − | font-family:Arial;color:black'> Brought
| + | |
| − | volume up to 500 mL with sterile H2O</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal style='text-indent:.5in'><span style='font-size:11.0pt;
| + | |
| − | font-family:Arial;color:black'> Autoclaved
| + | |
| − | to sterilize</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-family:"Times New Roman"'> </span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'>10/8/16</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'>Transformations</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'> Transformed
| + | |
| − | TEF2 + OXTR into pSB1K3</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'> Transformed
| + | |
| − | PGK1 + OXTR into pSB1K3</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'> Note:
| + | |
| − | see Benchling entry for protocol details</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-family:"Times New Roman"'> </span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'>Made
| + | |
| − | overnight cultures of:</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'> TEF2
| + | |
| − | in PSBIC3</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'> KOZAK
| + | |
| − | in PSBIC3</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'> mCherry
| + | |
| − | in pSB1K3</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'> ADHI
| + | |
| − | terminator in pSB1K3</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-family:"Times New Roman"'> </span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'> In
| + | |
| − | 3 mL of LB and 3 </span><span style='font-size:11.0pt;font-family:"Cambria Math";
| + | |
| − | color:black'>𝜇</span><span style='font-size:11.0pt;font-family:Arial;
| + | |
| − | color:black'>L of Chlor</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal style='margin-bottom:12.0pt'><span style='font-family:"Times New Roman"'> </span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'>10/9/16
| + | |
| − | CK </span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'>Minipreps</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'> ADHI
| + | |
| − | terminator BBa_K801812 (1G in Nunc box)</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'> TEF2
| + | |
| − | promoter BBa_K165037 (2G in Nunc box)</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'> KOZAK
| + | |
| − | BBa_K165002 (1F in Nunc box)</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'> mCherry
| + | |
| − | BBa_K165004 (3G in Nunc box)</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-family:"Times New Roman"'> </span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'> Heated
| + | |
| − | elution buffer to 55 degrees C</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-family:"Times New Roman"'> </span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'>Restriction
| + | |
| − | Digests</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'> ADHI</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'> TEF2</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'> mCherry</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-family:"Times New Roman"'> </span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'> 10
| + | |
| − | </span><span style='font-size:11.0pt;font-family:"Cambria Math";color:black'>𝜇</span><span
| + | |
| − | style='font-size:11.0pt;font-family:Arial;color:black'>L DNA</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'> 2
| + | |
| − | </span><span style='font-size:11.0pt;font-family:"Cambria Math";color:black'>𝜇</span><span
| + | |
| − | style='font-size:11.0pt;font-family:Arial;color:black'>L FD green buffer</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'> 1
| + | |
| − | </span><span style='font-size:11.0pt;font-family:"Cambria Math";color:black'>𝜇</span><span
| + | |
| − | style='font-size:11.0pt;font-family:Arial;color:black'>L NotI</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'> <u>7
| + | |
| − | </u></span><u><span style='font-size:11.0pt;font-family:"Cambria Math";
| + | |
| − | color:black'>𝜇</span></u><u><span style='font-size:11.0pt;font-family:
| + | |
| − | Arial;color:black'>L </span></u><span style='font-size:11.0pt;font-family:Arial;
| + | |
| − | color:black'>H2O</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'> 20
| + | |
| − | </span><span style='font-size:11.0pt;font-family:"Cambria Math";color:black'>𝜇</span><span
| + | |
| − | style='font-size:11.0pt;font-family:Arial;color:black'>L total</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-family:"Times New Roman"'> </span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'> Incubated
| + | |
| − | at 37 degrees C for 30 minutes</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'> Heat
| + | |
| − | inactivated at 80 degrees C</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'> Samples
| + | |
| − | arranged in gel:</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'> Ladder
| + | |
| − | - ADHI - TEF2 - mCherry</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal style='text-indent:.5in'><span style='font-size:11.0pt;
| + | |
| − | font-family:Arial;color:black'>Gel ran for 15 minutes</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-family:"Times New Roman"'> </span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal style='text-indent:.5in'><span style='font-size:11.0pt;
| + | |
| − | font-family:Arial;color:black'>Results: ADHI successful (</span><span
| + | |
| − | style='font-size:11.0pt;font-family:Arial;color:#4A86E8'>Yay!</span><span
| + | |
| − | style='font-size:11.0pt;font-family:Arial;color:black'>), TEF2 failed </span><span
| + | |
| − | style='font-size:11.0pt;font-family:Arial;color:#4A86E8'>:(</span><span
| + | |
| − | style='font-size:11.0pt;font-family:Arial;color:black'>, mCherry successful (</span><span
| + | |
| − | style='font-size:11.0pt;font-family:Arial;color:#4A86E8'>woot!</span><span
| + | |
| − | style='font-size:11.0pt;font-family:Arial;color:black'>)</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal style='text-indent:.5in'><span style='font-size:11.0pt;
| + | |
| − | font-family:Arial;color:black'> Saw
| + | |
| − | appropriate bands for ADHI and mCherry, no second band for TEF2</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-family:"Times New Roman"'> </span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'>10/10/16
| + | |
| − | CK</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'> Made
| + | |
| − | Gal 1 colonies</span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal style='margin-bottom:12.0pt'><span style='font-family:"Times New Roman"'> </span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal><span style='font-size:11.0pt;font-family:Arial;color:black'> </span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal style='margin-bottom:12.0pt'><span style='font-family:"Times New Roman"'> </span></p>
| + | |
| − | | + | |
| − | <p class=MsoNormal> </p>
| + | |
| − | | + | |
| − | </div>
| + | |
| − | | + | |
| − | </article> | + | |
| − | | + | |
| | </p> | | </p> |
| | </body> | | </body> |
