Difference between revisions of "Team:UCLA/Notebook"

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       </div>
 
       </div>
 
     </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>Clone and transform</h5>
 +
        <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
 +
              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.
 +
          </p>
 +
          <br/>
 +
          <p dir="ltr">
 +
              Made plates
 +
          </p>
 +
          <ul>
 +
              <li dir="ltr">
 +
                  <p dir="ltr">
 +
                      12.5g LB
 +
                  </p>
 +
              </li>
 +
              <li dir="ltr">
 +
                  <p dir="ltr">
 +
                      7.5 Bacto Agar
 +
                  </p>
 +
              </li>
 +
              <li dir="ltr">
 +
                  <p dir="ltr">
 +
                      500mL dH2O
 +
                  </p>
 +
              </li>
 +
              <li dir="ltr">
 +
                  <p dir="ltr">
 +
                      500uL Kanomycin (50mg/mL)
 +
                  </p>
 +
              </li>
 +
          </ul>
 +
          <br/>
 +
          <ul>
 +
              <li dir="ltr">
 +
                  <p dir="ltr">
 +
                      20 minutes autoclave
 +
                  </p>
 +
              </li>
 +
              <li dir="ltr">
 +
                  <p dir="ltr">
 +
                      4 hours total time to solidify
 +
                  </p>
 +
              </li>
 +
          </ul>
 +
          <p dir="ltr">
 +
              =&gt; 32 plates total
 +
          </p>
 +
          <br/>
 +
          <p dir="ltr">
 +
              Transformation
 +
          </p>
 +
          <ul>
 +
              <li dir="ltr">
 +
                  <p dir="ltr">
 +
                      30uL iGEM electrocompetent cells
 +
                  </p>
 +
              </li>
 +
              <li dir="ltr">
 +
                  <p dir="ltr">
 +
                      1uL O333 vector 1:100 dilution
 +
                  </p>
 +
              </li>
 +
              <li dir="ltr">
 +
                  <p dir="ltr">
 +
                      970uL SOC save
 +
                  </p>
 +
              </li>
 +
          </ul>
 +
          <br/>
 +
          <ul>
 +
              <li dir="ltr">
 +
                  <p dir="ltr">
 +
                      45 minutes incubation @ 800rpm/37C
 +
                  </p>
 +
              </li>
 +
              <li dir="ltr">
 +
                  <p dir="ltr">
 +
                      1 and 1:10 dilution plating
 +
                  </p>
 +
              </li>
 +
          </ul>
 +
          <p dir="ltr">
 +
              =&gt; 3 transformations total
 +
          </p>
 +
          <p dir="ltr">
 +
              =&gt; arc time: 5.5s, 5.6s, 5.6s
 +
          </p>
 +
          <br/>
 +
          <p dir="ltr">
 +
              **Plates in 37C inoculation from 6:40pm
 +
          </p>
 +
          <br/>
 +
          <p dir="ltr">
 +
              Conclusions: Transformations had a consistent arc time. Tomorrow morning plates will be checked for colonies and grown.
 +
          </p></p>
 +
      </div>
 +
    </div>
 +
 +
  
 
<!-- PCAGE NEW DAY BEFORE THIS LINE --!>
 
<!-- PCAGE NEW DAY BEFORE THIS LINE --!>

Revision as of 19:23, 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

Jun

24

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

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

June

14

Clone and transform

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.