Difference between revisions of "Team:UCLA/Notebook"

Line 105: Line 105:
 
         <ul> <b>
 
         <ul> <b>
 
           <li> 98C -- 10s</li>
 
           <li> 98C -- 10s</li>
−
           <li> 66C -- 30s</li>
+
           <li> TmC -- 30s</li>
−
           <li> 72C -- 140s</li>
+
           <li> 72C -- ext time</li>
 
         </b> </ul>  
 
         </b> </ul>  
 
         <ul>
 
         <ul>
 
           <li> 72C -- 2min</li>  
 
           <li> 72C -- 2min</li>  
 
         </ul>
 
         </ul>
−
<p> 28 cycles </p>
+
        <p> 28 cycles </p>
 
    
 
    
 +
      </div>
 +
    </div>
 +
 +
    <div class="row day-entry">
 +
      <div class="columns small-2 small-offset-1 day-date">
 +
<span>Jun</span>
 +
<p>30</p>
 +
      </div>
 +
      <div class="columns small-9">
 +
<h5>Troubleshooting PCR of Gibson fragments</h5>
 +
<ul>
 +
          <li>try using KAPA Hifi instead of Q5</li>
 +
          <li>add more template gDNA (1ng, 10ng, 35ng, 70ng)</li>
 +
        <p> 25uL rxn: </p>
 +
        <ul>
 +
          <li>0.75 uL F primer</li>
 +
          <li>0.75 uL R primer</li>
 +
          <li>12.5 uL KAPA Hifi 2X MM</li>
 +
          <li>dH2O up to 25 uL</li>
 +
        </ul>
 +
        <p> Fragments: </p>
 +
        <ul>
 +
          <li>EBL1 in pSB1C3 frag 2 (Tm = 66C, ext time = 140s)</li>
 +
          <li>EBL2 in pSB1C3 frag 2 (Tm = 65C, ext time = 88s)</li>
 +
          <li>EBL2 in pSB1C3 frag 3 (Tm = 65C, ext time = 180s)</li>
 +
        </ul>
 +
          <p> Cycling conditions: </p>
 +
        <ul>
 +
          <li> 98C -- 30s</li>
 +
        </ul>
 +
        <ul> <b>
 +
          <li> 98C -- 10s</li>
 +
          <li> TmC -- 30s</li>
 +
          <li> 72C -- ext time</li>
 +
        </b> </ul>
 +
        <ul>
 +
          <li> 72C -- 2min</li>
 +
        </ul>
 +
        <p> 28 cycles </p>
 
       </div>
 
       </div>
 
     </div>
 
     </div>

Revision as of 01:07, 10 August 2016

JOURNAL

Projects

CDI

Protein Cages

Week

1

Construction of ATCC 13048 Loci 2 in pSB1C3

Isolation of template DNA from EC93, EC869, and ATCC 13048; PCR of Gibson fragments for EBL2 in pSB1C3 construct

Jun

27

Inoculation of strains obtained from Dr. Christopher Hayes

Inoculated overnight cultures of:

  • EPI100 w/ EC93 cosmid
  • X90 w/ EC869 cosmid
  • ATCC 13048

Conditions:

  • 10mL LB
  • 10uL ampicillin
  • 37 C for 16h shaking
Jun

28

Isolation of template DNA

Miniprep of EC93 and EC869

  • EC93 #1: 145.42 ng/μL
  • EC93 #2: 198.85 ng/μL
  • EC869 #1: 90.81 ng/μL
  • EC869 #2: 45.95 ng/μL

gDNA isolation of ATCC 13048

  • 34.87 ng/μL

Gibson primers designed for EBL1 and EBL2 toxin switched constructs in pSB1C3 and pSC101

Jun

29

PCR of Gibson fragments for EBL1 and EBL2 in pSB1C3

50uL rxn:

  • 2.5 uL F primer
  • 2.5 uL R primer
  • 25 uL Q5 2X MM
  • 19 uL dH2O

Fragments:

  • EBL1 in pSB1C3 frag 2
  • EBL2 in pSB1C3 frag 2
  • EBL2 in pSB1C3 frag 3

Cycling conditions:

  • 98C -- 30s
  • 98C -- 10s
  • TmC -- 30s
  • 72C -- ext time
    •
  • 72C -- 2min

28 cycles

Jun

30

Troubleshooting PCR of Gibson fragments
  • try using KAPA Hifi instead of Q5
  • add more template gDNA (1ng, 10ng, 35ng, 70ng)

    • 25uL rxn:

    • 0.75 uL F primer
    • 0.75 uL R primer
    • 12.5 uL KAPA Hifi 2X MM
    • dH2O up to 25 uL

    Fragments:

    • EBL1 in pSB1C3 frag 2 (Tm = 66C, ext time = 140s)
    • EBL2 in pSB1C3 frag 2 (Tm = 65C, ext time = 88s)
    • EBL2 in pSB1C3 frag 3 (Tm = 65C, ext time = 180s)

    Cycling conditions:

    • 98C -- 30s
    • 98C -- 10s
    • TmC -- 30s
    • 72C -- ext time
      •
    • 72C -- 2min

    28 cycles

Week

1

Title For The Week

Description blah blah blah

Jun

24

Title for the day (e.g. "Cloning Mutants")

Today we did cloning. fuck cloning. blah blah blah.