Week Nine

Monday, 8/11/14

• 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.

• Continued vacufuging LCMS acetonitrile pTG004-FLAG samples.

• Miniprepped pMB001, 2, & 5 liquid cultures. Shipped them out for sequencing.
• Resuspended colonies for pMB003, 4, & 6 and grew up overnight cultures of them.

• 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.

Tuesday, 8/12/14

• Miniprepped yesterday's 2 liquid cultures of A2 and shipped them for sequencing

• Continued vacufuging LC/MS acetonitrile samples (lam-noFLAG)

• 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.

Wednesday, 8/13/14

• PCRed out backbones for pTG002, pTG003, & pTG006.
• DpnI digested and then purified PCR products.
• Ran a gel to confirm isolation of backbones from above procedures.
• Gibson assembly using constructed backbones and appropriate gblocks.
• Gibson assembly products were transformed into JM109 cells. 2 transformations were performed for each product: one using 3 μL of assembly product and the other using 11 μL of assembly product.
• Transformations werew plated on CARB plates & incubated overnight.

• Innoculated LB-CARB liquid culture with DH5α cells expressing pTG005 (fsr system with FLAG still included)

• Miniprepped pMB003, pMB004, & pMB006 liquid cultures. Sent samples out for sequencing.
• Resuspended pMB005 colonies and ran colony PCR.

• 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.

Thursday, 8/14/14

• Ran colony PCR on yesterday's transformations.
• Colony PCR products were run on a gel.
• 8 LB-CARB liquid cultures were prepared and incubated overnight.

• Picked more colonies from A2 transformation last week and ran colony PCR on them.
• Ran a gel on the colony PCR products.
• Set up 4 LB-CARB liquid cultures and incubated them overnight.

• Set up 0 nM, 250 nM, & 500 nM aTc inductions of pTG005-expressing cells (fsr-FLAG) in MOPS media. 5 μL aliquots of last night's liquid culture was used to start the cultures in MOPS.

• 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.

Friday, 8/15/14

• Miniprepped 3 of the 8 LB liquid cultures from last night.
• Shipped samples out for sequencing.

• Miniprepped 3 of the 4 LB liquid cultures from last night.
• Shipped samples out for sequencing.

• Started LB liquid culture of DH5α cells expressing pAA001 as a negative control in the LC/MS runs.
• aTc-induced MOPS liquid cultures from yesterday were spun down, and then the supernatant was loaded into C18 columns, washed with 10% acetonitrile, and then eluted with 60% acetonitrile.
• The pTG005 acetonitrile elutates were then left to evaporate in the fume hood overnight.
• On Saturday, we began vacufuging the fsr-FLAG LC/MS acetonitrile samples.
• On Saturday, we also set up 0 nM, 250 nM, & 500 nM aTc inductions of pAA001-expressing cells (negative control in LC/MS) in MOPS media. 5 μL aliquots of last night's liquid culture was used to start the cultures.
• On Sunday, we spun down the aTc-induced MOPS liquid cultures of pAA001 cells, and then loaded the supernatant into C18 columns, washed it with 10% acetonitirle, and then eluted it with 60% acetonitrile.
• pAA001 acetonitrile samples were then left to evaporate in the fume hood overnight.

• Attempted to PCR-out the 3xFLAG from pTG005.
• DpnI digested and PCR-purified the pTG005 PCR product.
• Ran round-the-horn ligation on the purified PCR product.
• Transformed round-the-horn ligation product into DH5α

• 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.