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
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Week Nine |
Monday, 8/11/14
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Export Systems
Attempt to create a negative control for Western blotting:
- Ran colony PCR on colonies of A2 construct (pTet-GFP). Annealing temperature was 53°C. Colonies were overloaded and smeared the lanes.
- Reran colony PCR on A2 colonies. Annealing temperature was 51°C. 2 presumed successes.
- Set up 2 liquid cultures using the 2 colonies proposed to have A2.
LCMS analysis of lam system:
- Continued vacufuging LCMS acetonitrile pTG004-FLAG samples.
lamBCDA & fsrABC Reception Systems
- Miniprepped pMB001, 2, & 5 liquid cultures. Shipped them out for sequencing.
- Resuspended colonies for pMB003, 4, & 6 and grew up overnight cultures of them.
agrBCDA Reception System and Combinatorial Promoters
agrBCDA system:
- Set up liquid cultures of pAA009 & pAA010 colonies from overnight.
- Ran colony PCR on pAA009 & pAA010 colonies, adding DMSO & with annealing temperature of 51°C. This yielded bright bands at correct length for 2 of the pAA010 colonies and some of the pAA009 colonies.
- PCR-purified the pAA010 colony PCR products & shipped them for sequencing.
- Miniprepped pAA009 liquid cultures & sent samples for sequencing.
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Tuesday, 8/12/14
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Export Systems
agrBCDA Reception System and Combinatorial Promoters
- Analyzed sequencing data, indicating that 2 of the pAA010 Gibson colonies contain the correct sequence.
- Miniprepped liquid cultures of those colonies.
- Since pAA009 construction was a failure, we ran another Gibson assembly constructing pAA009.
- New Gibson assemblies were transformed into DH5α-Z1.
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Wednesday, 8/13/14
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Export Systems
lamBCDA & fsrABC Reception Systems
- Miniprepped pMB003, pMB004, & pMB006 liquid cultures. Sent samples out for sequencing.
- Resuspended pMB005 colonies and ran colony PCR.
agrBCDA Reception System and Combinatorial Promoters
- Ran colony PCR on colonies from the pAA009 Gibson assembly transformation yesterday.
- Ran a gel of the colony PCR products.
- PCR-purified promising PCR products and shipped them for sequencing.
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Thursday, 8/14/14
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Export Systems
lamBCDA & fsrABC Reception Systems
agrBCDA Reception System and Combinatorial Promoters
- Analyzed sequencing results from yesterday. Bad results for all.
- Some colonies that produced bands of right length from yesterday's colPCR weren't sequenced, so those colonies were miniprepped and then shipped for sequencing.
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Friday, 8/15/14
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Export Systems
lamBCDA & fsrABC Reception Systems
agrBCDA Reception System and Combinatorial Promoters
- Analyzed sequencing results from yesterday's minipreps. Failed again.
- Conducted Round-the-horn PCR using sequence-verified pAA010 plasmids to attempt to construct pAA009.
- DpnI-digested for 1.5 hours and then PCR-purified PCR products.
- QuickLigation reaction on purified PCR product. Ligation product was then transformed into JM109.
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