Difference between revisions of "Team:TP-CC San Diego/LabBook"

 
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<h2 class = "main-title"> Lab Notebook </h2>
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<h2 class = "main-title fade-in"> Lab Notebook </h2>
  
 
<div class = "text-wrapper">
 
<div class = "text-wrapper">
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<center><a href="https://static.igem.org/mediawiki/2017/8/81/T--TP-CC_San_Diego--labnotebook.pdf">Click Here for the full notebook PDF</a></center>
+
<center><a href="https://2017.igem.org/Team:TP-CC_San_Diego/Protocols">Click Here to Refer to Protocols Page</a></center>
 
     <h3 class = "day"> DAY 1 </h3>
 
     <h3 class = "day"> DAY 1 </h3>
 
     <div class = "description">  
 
     <div class = "description">  
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       </p>
 
       </p>
 
       <ul>  
 
       <ul>  
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           <li>Primers arrived</li>
+
           <li>Primers arrived and diluted according to instructions</li>
 
       </ul>
 
       </ul>
 
   </div>
 
   </div>
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         </p>
 
         </p>
 
         <ul>
 
         <ul>
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             <li>Received oligos </li>
+
             <li>Received oligos and diluted according to instruction.</li>
 
         </ul>
 
         </ul>
 
     </div>
 
     </div>
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         </ul>
 
         </ul>
 
     </div>
 
     </div>
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 +
<h3 class = "day"> DAY 14 </h3>
 +
 
 +
    <div class = "description">
 +
        <p>
 +
            <b>SUMMARY of August 21st: </b>
 +
        </p>
 +
        <ul>
 +
            <li>seed ~1.2 x 107 293T cells in a 10 cm dish </li>
 +
        </ul>
 +
    </div>
  
 +
 +
 +
 +
<h3 class = "day"> DAY 15 </h3>
 +
 
 +
    <div class = "description">
 +
        <p>
 +
            <b>SUMMARY of August 17th: </b>
 +
        </p>
 +
        <ul>
 +
            <li>Prepared the transfection complex. </li>
 +
        </ul>
 +
    </div>
 +
 +
 +
<h3 class = "day"> DAY 16 </h3>
 +
 
 +
    <div class = "description">
 +
        <p>
 +
            <b>SUMMARY of August 23rd: </b>
 +
        </p>
 +
        <ul>
 +
            <li>Removed the transfection medium from cells </li>
 +
            <li>Miniprep plasmids and measure concentration </li>
 +
        </ul>
 +
    </div>
 +
 +
 +
<h3 class = "day"> DAY 17 </h3>
 +
 
 +
    <div class = "description">
 +
        <p>
 +
            <b>SUMMARY of August 24th: </b>
 +
        </p>
 +
        <ul>
 +
            <li>Harvest cells </li>
 +
        </ul>
 +
    </div>
 +
 +
 +
<h3 class = "day"> DAY 18 </h3>
 +
 
 +
    <div class = "description">
 +
        <p>
 +
            <b>SUMMARY of August 25th: </b>
 +
        </p>
 +
        <ul>
 +
            <li> Plate target cells, incubate overnight </li>
 +
        </ul>
 +
    </div>
 +
 +
 +
 +
<h3 class = "day"> DAY 19 </h3>
 +
 
 +
    <div class = "description">
 +
        <p>
 +
            <b>SUMMARY of August 26th: </b>
 +
        </p>
 +
        <ul>
 +
            <li> Remove the cell culture medium and replace with fresh medium, incubate overnight </li>
 +
        </ul>
 +
    </div>
 +
 +
<h3 class = "day"> DAY 20 </h3>
 +
 
 +
    <div class = "description">
 +
        <p>
 +
            <b>SUMMARY of August 27th: </b>
 +
        </p>
 +
        <ul>
 +
            <li> Split cells 1:3 and incubate for 48 hours </li>
 +
        </ul>
 +
    </div>
 +
 +
 +
<h3 class = "day"> DAY 21 </h3>
 +
 
 +
    <div class = "description">
 +
        <p>
 +
            <b>SUMMARY of August 29th: </b>
 +
        </p>
 +
        <ul>
 +
            <li> Infected cells are selected for stable expression </li>
 +
            <li> Continue monitoring cells  </li>
 +
        </ul>
 +
    </div>
 +
 +
 +
<h3 class = "day"> DAY 22 </h3>
 +
 
 +
    <div class = "description">
 +
        <p>
 +
            <b>SUMMARY of October 21st: </b>
 +
        </p>
 +
        <ul>
 +
            <li> Digest guideRNA using restriction enzymes out of TLCV2 vector </li>
 +
            <li> Run 1% agarose gel and cut out insert </li>
 +
            <li> Gel purify insert and vector(pSB1C3) given by iGEM </li>
 +
            <li> Ligation of guide RNA insert and pSB1C3 vector </li>
 +
            <li> Transformation of plasmid for part submission</li>
 +
        </ul>
 +
    </div>
 +
 +
<h3 class = "day"> DAY 23 </h3>
 +
 
 +
    <div class = "description">
 +
        <p>
 +
            <b>SUMMARY of October 22nd: </b>
 +
        </p>
 +
        <ul>
 +
            <li> Inoculated plasmid for part submission </li>
 +
        </ul>
 +
    </div>
 +
 +
 +
<h3 class = "day"> DAY 24 </h3>
 +
 
 +
    <div class = "description">
 +
        <p>
 +
            <b>SUMMARY of October 23st: </b>
 +
        </p>
 +
        <ul>
 +
            <li> Miniprep plasmids and measure concentration </li>
 +
        </ul>
 +
    </div>
 +
 +
 +
</div>
  
 
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Latest revision as of 02:02, 2 November 2017

Lab Notebook

Click Here to Refer to Protocols Page

DAY 1

SUMMARY of July 17th:

  • DNA extraction from GBM39 cells
  • Agarose gel electrophoresis to confirm DNA

DAY 2

SUMMARY of July 18th:

  • qPCR primer design and order

DAY 3

SUMMARY of July 21:

  • Primers arrived and diluted according to instructions

DAY 4

SUMMARY of August 1st:

  • qPCR for EGFR gene count and guide RNA design; placed order

DAY 5

SUMMARY of August 2nd:

  • Received oligos and diluted according to instruction.

DAY 6

SUMMARY of August 7th:

  • Digestion of lentiviral CRISPR plasmid
  • Gel purification digested plasmid using QIAquick Gel Extraction Kit and eluted in buffer EB
  • There should be two bands on the gel, one at 2kb which should not be purified. Purify only the larger band

DAY 7

SUMMARY of August 9th:

  • Anneal oligo pairs
  • Ligation and transformation

DAY 8

SUMMARY of August 10th:

  • No colonies grew on carb antibiotic plates

DAY 9

SUMMARY of August 11th:

  • Used same,remaining ligation reaction and transformed again onto carb antibiotic plates

DAY 10

SUMMARY of August 14th:

  • Inoculate comp cells

DAY 11

SUMMARY of August 15th:

  • Miniprep gRNA plasmids and sent in for sequencing
  • Start interlab study
  • Ludox Calibration
  • Fluorescein Dilution
  • Transform plasmids from plate 7 in kit provided by IGEM for interlab study

DAY 12

SUMMARY of August 16th:

  • Received sequencing results and all plasmids have the gRNA
  • Inoculate plasmids for interlab study

DAY 13

SUMMARY of August 17th:

  • Interlab cell measurement

DAY 14

SUMMARY of August 21st:

  • seed ~1.2 x 107 293T cells in a 10 cm dish

DAY 15

SUMMARY of August 17th:

  • Prepared the transfection complex.

DAY 16

SUMMARY of August 23rd:

  • Removed the transfection medium from cells
  • Miniprep plasmids and measure concentration

DAY 17

SUMMARY of August 24th:

  • Harvest cells

DAY 18

SUMMARY of August 25th:

  • Plate target cells, incubate overnight

DAY 19

SUMMARY of August 26th:

  • Remove the cell culture medium and replace with fresh medium, incubate overnight

DAY 20

SUMMARY of August 27th:

  • Split cells 1:3 and incubate for 48 hours

DAY 21

SUMMARY of August 29th:

  • Infected cells are selected for stable expression
  • Continue monitoring cells

DAY 22

SUMMARY of October 21st:

  • Digest guideRNA using restriction enzymes out of TLCV2 vector
  • Run 1% agarose gel and cut out insert
  • Gel purify insert and vector(pSB1C3) given by iGEM
  • Ligation of guide RNA insert and pSB1C3 vector
  • Transformation of plasmid for part submission

DAY 23

SUMMARY of October 22nd:

  • Inoculated plasmid for part submission

DAY 24

SUMMARY of October 23st:

  • Miniprep plasmids and measure concentration