Laboratory Notebook

09-07-2012 to 15-07-2012

PCR of SST and FFT: When the primers arrived, 1-SST and 1-FFT were amplified with PCR(*polymerase chain reaction*) and isolated using gel electrophoresis.
The gel showed clear bands near 1900bp, which is the expected lengths of our genes of interest.
The PCR products from the gel were cut out and extracted using a PCR-extraction kit.

The results from two succesful FFT gel bands on Nanodrop:
345,5 ng/uL and 436,2 ng/uL

We did not get any bands for SST and made another PCR at different temperatures. We did get 2 weak bands for SST.
The results from Nanodrop:
15,5ng/uL and 35,7ng/uL

The results were not satisfying, but decent enough to move on to make extension PCR with the iGEM prefix and suffix overhangs in order to introduce the standardized restriction sites.

We did not get immidiate good results, determined by Nanodrop analysis, so we experimented with different temperatures for the PCR annealing step. After some days of trial and error, we had decent nanodrop results, >10ng/uL, and we moved on to ligation and transformation.

We used the pJET1.2/BLUNT vector as transformation plasmid, since the labatory staff had good experience with this plasmid. It carries the ampicillin resistance and an EcoRV(blunt cut) restriction site within a toxin coding sequence with its own promoter and terminator.

The blunt ligation was immidiately followed by transformation and the cultures were incubated overnight on ampicilin agar media plates.

We had succes with 10 1-FFT colonies, but no luck with 1-SST, so we had to do another ligation and transformation. FFT colonies were transferred to liquid ampicillin containing medium and incubated overnight with SST ampicillin agar plates.