Team:Caltech/week6

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<b>Export Systems</b>
<b>Export Systems</b>
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3 of our 5 constructs were not properly cloned, so we are going to redo them:
3 of our 5 constructs were not properly cloned, so we are going to redo them:
<ul><li>PCRed out backbones for pTG002, pTG003, and pTG006 with overhangs</li>
<ul><li>PCRed out backbones for pTG002, pTG003, and pTG006 with overhangs</li>
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<b>agrBCDA Reception System and Combinatorial Promoters</b>
<b>agrBCDA Reception System and Combinatorial Promoters</b>
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For the agrBCDA System, we troubleshot our problems creating the pAA009/10 vector backbone:
For the agrBCDA System, we troubleshot our problems creating the pAA009/10 vector backbone:
<ul><li>Ran a gel of the PCR product created on Friday (pAA009/10 backbone). Results were negative.</li>
<ul><li>Ran a gel of the PCR product created on Friday (pAA009/10 backbone). Results were negative.</li>

Revision as of 18:45, 22 July 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 Six

Monday, 7/21/14

 Export Systems
3 of our 5 constructs were not properly cloned, so we are going to redo them:
  • PCRed out backbones for pTG002, pTG003, and pTG006 with overhangs
  • PCR product was then DpnI digested and PCR-purified. A gel was run with processed and unprocessed product to verify backbone creation
  • Gibson assembly of pTG002, pTG003, and pTG006 (pTG006 had 2 reactions: 1 with the backbone created last week, since it had been verified to work for cloning pTG005, and another with backbone created today)
  • Assemblies were transformed into JM109, plated on carbenicillin plates, and incubated overnight
lamBCDA & fsrABC Reception Systems
  • 4 cultures of JM109 were transformed, plated on Kan + Cm plates, and incubated overnight at 37°C:
    1. pMB001 and pMB003
    2. pMB002 and pMB003
    3. pMB004 and pMB006
    4. pMB005 and pMB006
agrBCDA Reception System and Combinatorial Promoters
For the agrBCDA System, we troubleshot our problems creating the pAA009/10 vector backbone:
  • Ran a gel of the PCR product created on Friday (pAA009/10 backbone). Results were negative.
  • Tried PCR again with lower annealing temperatures [(what temps?)]. Results were negative again.
For the combinatorial promoters:
  • Set up overnight culture of jwAA001 strain to do more experiments tomorrow

Tuesday, 7/22/14

Wednesday, 7/23/14

Thursday, 7/24/14

Friday, 7/25/14

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