Difference between revisions of "Team:UCLA/Notebook"

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       <div class="columns small-2 small-offset-1 day-date">
 
       <div class="columns small-2 small-offset-1 day-date">
 
         <span>June</span>
 
         <span>June</span>
−
         <p>14</p>
+
         <p>13</p>
 
       </div>
 
       </div>
 
       <div class="columns small-9">
 
       <div class="columns small-9">
−
         <h5>Clone and transform</h5>
+
         <h5>Plate Making and Transformation of O3-33 WT</h5>
 
         <p><p dir="ltr">
 
         <p><p dir="ltr">
 
               Goals for today: We have a vector of O333 from Neil King contained in a pET vector, the goal for today is to transform this vector, grow it tomorrow and
 
               Goals for today: We have a vector of O333 from Neil King contained in a pET vector, the goal for today is to transform this vector, grow it tomorrow and
Line 286: Line 286:
 
           <p dir="ltr">
 
           <p dir="ltr">
 
               Conclusions: Transformations had a consistent arc time. Tomorrow morning plates will be checked for colonies and grown.
 
               Conclusions: Transformations had a consistent arc time. Tomorrow morning plates will be checked for colonies and grown.
 +
          </p></p>
 +
      </div>
 +
    </div>
 +
 +
 +
    <div class="row day-entry">
 +
      <div class="columns small-2 small-offset-1 day-date">
 +
        <span>June</span>
 +
        <p>14</p>
 +
      </div>
 +
      <div class="columns small-9">
 +
        <h5>Transformed O3-33 WT</h5>
 +
        <p><h1 dir="ltr">
 +
              June 14th Lab Journal
 +
          </h1>
 +
          <p dir="ltr">
 +
              <strong id="docs-internal-guid-955c1d56-75eb-5ceb-fe34-d30c89713871">
 +
                  <br/>
 +
              </strong>
 +
          </p>
 +
          <p dir="ltr">
 +
              Recap from previous day: No colonies were shown from any of the transformations. Competent cells may have been of low quality (relatively long arc times).
 +
          </p>
 +
          <p dir="ltr">
 +
              <strong>
 +
                  <br/>
 +
              </strong>
 +
          </p>
 +
          <p dir="ltr">
 +
              Goals: Today, commercial electro-competent cells are going to be transformed, and different concentrations of DNA will be tested for transformation as
 +
              well.
 +
          </p>
 +
          <p dir="ltr">
 +
              <strong>
 +
                  <br/>
 +
              </strong>
 +
          </p>
 +
          <p dir="ltr">
 +
              Transformation:
 +
          </p>
 +
          <ul>
 +
              <li dir="ltr">
 +
                  <p dir="ltr">
 +
                      3x 25uL commercial electrocompetent cells
 +
                  </p>
 +
              </li>
 +
              <li dir="ltr">
 +
                  <p dir="ltr">
 +
                      1uL of 1:5, 1:50, 1:100 serial dilutions of plasmid from Neil King
 +
                  </p>
 +
              </li>
 +
              <li dir="ltr">
 +
                  <p dir="ltr">
 +
                      970uL SOC save
 +
                  </p>
 +
              </li>
 +
          </ul>
 +
          <p dir="ltr">
 +
              <strong>
 +
                  <br/>
 +
              </strong>
 +
          </p>
 +
          <ul>
 +
              <li dir="ltr">
 +
                  <p dir="ltr">
 +
                      60 minutes save (800rpm @ 37C), then additional 50 minutes at 37C incubator (no shaking) as I was waiting for plating beads to be autoclaved and
 +
                      cooled
 +
                  </p>
 +
              </li>
 +
              <li dir="ltr">
 +
                  <p dir="ltr">
 +
                      1 and 1:10 dilution platings
 +
                  </p>
 +
              </li>
 +
          </ul>
 +
          <p dir="ltr">
 +
              =&gt; 1:5 DNA: 5.1s arc time
 +
          </p>
 +
          <p dir="ltr">
 +
              =&gt; 1:50 DNA: 5.0s arc time
 +
          </p>
 +
          <p dir="ltr">
 +
              =&gt; 1:100 DNA: 4.8s arc time
 +
          </p>
 +
          <p dir="ltr">
 +
              <strong>
 +
                  <br/>
 +
              </strong>
 +
          </p>
 +
          <p dir="ltr">
 +
              **Plates in 37C incubation from 4:10pm
 +
          </p>
 +
          <p dir="ltr">
 +
              <br/>
 +
              Conclusions: Same as yesterday, though today arc times were slightly better. Unfortunately, my timing was off for plating because I was waiting to
 +
              autoclave new beads (thanks for sharing, grad students!), so when I plated the transformants smelled pretty awful. Hopefully that doesn’t make too much of
 +
              a difference.
 +
              <br/>
 
           </p></p>
 
           </p></p>
 
       </div>
 
       </div>

Revision as of 19:32, 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

    ladder; EBL2 frag 2; EBL1 frag 2; EBL2 frag 3

Week

1

Title For The Week

Description blah blah blah

June

13

Plate Making and Transformation of O3-33 WT

Goals for today: We have a vector of O333 from Neil King contained in a pET vector, the goal for today is to transform this vector, grow it tomorrow and PCR it for a large number of copies to work with and creation of a biobrick. Primers will need to be designed including the T7 promoter to terminator, with iGEM prefix and suffix.


Made plates

  • 12.5g LB

  • 7.5 Bacto Agar

  • 500mL dH2O

  • 500uL Kanomycin (50mg/mL)


  • 20 minutes autoclave

  • 4 hours total time to solidify

=> 32 plates total


Transformation

  • 30uL iGEM electrocompetent cells

  • 1uL O333 vector 1:100 dilution

  • 970uL SOC save


  • 45 minutes incubation @ 800rpm/37C

  • 1 and 1:10 dilution plating

=> 3 transformations total

=> arc time: 5.5s, 5.6s, 5.6s


**Plates in 37C inoculation from 6:40pm


Conclusions: Transformations had a consistent arc time. Tomorrow morning plates will be checked for colonies and grown.

June

14

Transformed O3-33 WT

June 14th Lab Journal


Recap from previous day: No colonies were shown from any of the transformations. Competent cells may have been of low quality (relatively long arc times).


Goals: Today, commercial electro-competent cells are going to be transformed, and different concentrations of DNA will be tested for transformation as well.


Transformation:

  • 3x 25uL commercial electrocompetent cells

  • 1uL of 1:5, 1:50, 1:100 serial dilutions of plasmid from Neil King

  • 970uL SOC save


  • 60 minutes save (800rpm @ 37C), then additional 50 minutes at 37C incubator (no shaking) as I was waiting for plating beads to be autoclaved and cooled

  • 1 and 1:10 dilution platings

=> 1:5 DNA: 5.1s arc time

=> 1:50 DNA: 5.0s arc time

=> 1:100 DNA: 4.8s arc time


**Plates in 37C incubation from 4:10pm


Conclusions: Same as yesterday, though today arc times were slightly better. Unfortunately, my timing was off for plating because I was waiting to autoclave new beads (thanks for sharing, grad students!), so when I plated the transformants smelled pretty awful. Hopefully that doesn’t make too much of a difference.