AUGUST

Monday, August 4 - Friday, August 8

Tested PCR assembly products with Nanodrop machine to determine concentration of bases in each sample. Ran a negative control PCR for all four promoters, including all components except template DNA. Diluted PCR products tested on Nanodrop down to 220 base/mL, which was PFL2’s concentration, the lowest concentration of all samples. Ran a restriction enzyme digest on our PCR products, then ran an electrophoresis test on the negative controls. Decided to ligate PAR and PFL overnight; their short length may mean they require more time for ligation. Electrophoresis showed no bands for this ligation

Monday, August 11- Friday, August 15

Discussion with Dr. Peebles led to the plan to reconstruct PLR, PFL1, and PFL2. This was done according to the same protocol as earlier, with construction, PCR amplification, and electrophoresis check for product size accuracy all successful. Performed restriction enzyme digest of the three promoters and transformed pSB1C3 and pUC19 into competent cells. The three promoters were ligated overnight.

Monday, August 18 - Friday, August 22

Ligation products were transformed into competent cells. The transformation of pSB1C3 and pUC19 from last week did not result in any growth. Ran a 4 hour ligation on pSB1C3 in order to check if enzymes were working correctly. No bands were present in electrophoresis gel.Since there was no growth with the ligation products transformed into competent cells, we used a PCR product cleanup kit for our promoters. Electrophoresis of our products from this showed no bands.

Thursday, September 18th

After reviewing the work done over the summer, Dr. Peebles and I came to the conclusion that the most sensible choice would be to have the promoters sequenced in their entirety and move forward from then. The repeated failures of construction during the summer led to this conclusion. Promoter sequences were sent off to be sequenced in full, and we will continue this research next summer.

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