Team:Caltech/week7

From 2014.igem.org

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<b>agrBCDA Reception System and Combinatorial Promoters</b>
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<ul><li>Miniprepped liquid cultures grown last night and shipped samples for sequencing.</li>
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<b>agrBCDA Reception System and Combinatorial Promoters</b>
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<ul><li>Analyzed sequencing results:
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    <ul><li>pAA008 successfully constructed</li>
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        <li>Minipreps supposedly containing pAA009 & pAA010 instead contain pKS001 backbone template</li>
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    </ul></li>
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    <li>We redid PCR of the pAA009/010 backbone</li>
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    <li>DpnI digestion for 2 hours, followed by PCR purification. The purified product was then run on a gel.</li>
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Revision as of 23:07, 3 August 2014
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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 Seven

Monday, 7/28/14

 Export Systems
  • Ran colony PCR on pTG002/3/6-transformed JM109 cultures grown up over the weekend. Bad results for all of them
  • Created and ordered new primers that will hopefully avoid secondary-structure-formation when the exonuclease attacks the 5' end during Gibson assembly.
lamBCDA & fsrABC Reception Systems
  • Single-transformations run on each of the constructs used in the double-transformations:
    1. pMB001
    2. pMB002
    3. pMB003
    4. pMB004
    5. pMB005
    6. pMB006
agrBCDA Reception System and Combinatorial Promoters
  • Colonies were picked from pAA008 transformations incubated over the weekend, as well as the pAA009 & pAA010 transformations, which had been set up last Thursday.
  • Set up liquid cultures of the colonies to grow overnight in preparation for miniprep and sequencing tomorrow

Tuesday, 7/29/14

 Export Systems
  • Ran Gibson assembly products on a gel. Results inconclusive
  • Started 3 mL liquid cultures of bacteria (with pTG004 & pTG005) with varying levels of aTc induction:
    • 0 nM, 50 nM, 150 nM, 250 nM, 350 nM, & 450 nM
lamBCDA & fsrABC Reception Systems
  • Colony PCR was run on several of the colonies that emerged from the single-transformations last night
  • Gel was run, but wrong primers were used. New primers for sequencing/colPCR were ordered, and colPCR will be redone tomorrow.
agrBCDA Reception System and Combinatorial Promoters
  • Miniprepped liquid cultures grown last night and shipped samples for sequencing.

Wednesday, 7/30/14

 Export Systems
lamBCDA & fsrABC Reception Systems
agrBCDA Reception System and Combinatorial Promoters
  • Analyzed sequencing results:
    • pAA008 successfully constructed
    • Minipreps supposedly containing pAA009 & pAA010 instead contain pKS001 backbone template
  • We redid PCR of the pAA009/010 backbone
  • DpnI digestion for 2 hours, followed by PCR purification. The purified product was then run on a gel.

Thursday, 7/31/14

 Export Systems
lamBCDA & fsrABC Reception Systems
agrBCDA Reception System and Combinatorial Promoters

Friday, 8/1/14

 Export Systems
lamBCDA & fsrABC Reception Systems
agrBCDA Reception System and Combinatorial Promoters

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