Team:Caltech/week11

From 2014.igem.org

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Redo of cloning of agr system:
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<br>Redo of cloning of agr system:
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<ul><li>Ran PCR reactions to construct appropriate backbone for pTG002 & modified gblock insert for pTG002.</li>
 +
    <li>DpnI digested PCR product for pTG002 backbone.</li>
 +
    <li>PCR purified DpnI digested pTG002 backbone and pTG002 modified gblock insert PCR products.</li>
 +
    <li>Ran purified products on a gel.</li>
 +
    <li>Ran Gibson assembly reaction between pTG002-backbone & pTG002-insert.</li>
 +
    <li>Transformed Gibson reaction mix into JM109. Cells were then plated & incubated overnight.</li>
 +
</ul>
 +
De-FLAGging pTG005 for LC/MS analysis:
 +
<ul><li>Ran PCR reaction to exclude 3xFLAG from pTG005.</li>
 +
    <li>Ran DpnI digest and then PCR purification on PCR product.</li>
 +
    <li>Ran PCR product on a gel to verify creation of the appropriate DNA fragment.</li>
 +
    <li>Ran a round-the-horn ligation reaction on the purified PCR product.</li>
 +
    <li>Transformed the reaction mix into JM109. Cells were then plated & incubated overnight.</li>
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Latest revision as of 00:53, 15 September 2014
Home Team Official Team Profile Project Parts Modeling Notebook Safety Attributions
Notebook
Overview

Week 1

Week 2

Week 3

Week 4

Week 5

Week 6

Week 7

Week 8

Week 9

Week 10

Week 11

Week 12

Week 13

Week 14

Week 15

Week Eleven

Monday, 8/25/14

 lamBCDA & fsrABC Reception Systems
agrBCDA Reception System and Combinatorial Promoters

Tuesday, 8/26/14

 lamBCDA & fsrABC Reception Systems
agrBCDA Reception System and Combinatorial Promoters

Wednesday, 8/27/14

 lamBCDA & fsrABC Reception Systems
agrBCDA Reception System and Combinatorial Promoters

Thursday, 8/28/14

 lamBCDA & fsrABC Reception Systems
agrBCDA Reception System and Combinatorial Promoters
  • Re-ran a Gibson assembly of pAA009
  • Transformed Gibson products into JM109 cells

Friday, 8/29/14

 lamBCDA & fsrABC Reception Systems
agrBCDA Reception System and Combinatorial Promoters
  • Picked 10 Gibson colonies from yesterday
  • Ran colony PCR on re-suspended colonies, inoculated liquid cultures of picked colonies
  • Ran gel of colony PCR products, but no good bands were obtained

Saturday, 8/30/14

 Export Systems
Redo of cloning of agr system:
  • Ran PCR reactions to construct appropriate backbone for pTG002 & modified gblock insert for pTG002.
  • DpnI digested PCR product for pTG002 backbone.
  • PCR purified DpnI digested pTG002 backbone and pTG002 modified gblock insert PCR products.
  • Ran purified products on a gel.
  • Ran Gibson assembly reaction between pTG002-backbone & pTG002-insert.
  • Transformed Gibson reaction mix into JM109. Cells were then plated & incubated overnight.
De-FLAGging pTG005 for LC/MS analysis:
  • Ran PCR reaction to exclude 3xFLAG from pTG005.
  • Ran DpnI digest and then PCR purification on PCR product.
  • Ran PCR product on a gel to verify creation of the appropriate DNA fragment.
  • Ran a round-the-horn ligation reaction on the purified PCR product.
  • Transformed the reaction mix into JM109. Cells were then plated & incubated overnight.

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