Laboratory Notebook

09-07-2012 to 15-07-2012

PCR of SST and FFT

We did a polymerase chain reaction with two different settings; one where we used the recommended settings from the protocol and one where we specified the temperature for each cycle in the PCR which should be more optimal for the primers.
The genomic template we used was MCF7. The PCR samples were processed over night (O.N.).

Gel for amplification of FFT and SST

We did a gel on the PCR products from O.N. and got some usable bands from the FFT-gene.
We cut the band out of the gel a made a purification of it. We had some problems with the SST-gene and ended with no useable results.
We made a NanoDrop of the purified FFT-gene and got a concentration of -1.0ng/μl. We measured a NanoDrop on the PCR products from the FFT-gene before we ran a gel on it to compare with the purified FFT-gene. This gave us a concentration at 103,2ng/μl.
With this result we decided to repeat the purification of the FFT-gene.
We tried to optimise the concentration of the FFT-gene from the gel and ended up with 13,8ng/μl determined by the NanoDrop.
We decided to do a tranformation which was incubated O.N.
We prepared another PCR for the SST-gene where we adjusted variables, like raising the temperature to 63°C for the annealing-step and adjusted the extension time.
PCR was run overnight.

Gel electrophoresis of PCR products from 10-07-12 and preparation of PCR on FFT and SST DNA

We ran a gel electrophoresis the following day on the PCR products from the day before (the O.N. ones). After 1 hour at 100V we had fine separation of bands, and had very clear bands at the size where we had predicted our SST-gene to be: around 1900b.
The bands were cut out and purified using a PCR and gel clean-up.
The samples from the gel extraction were tested with Nanodrop which gave us uncertain results. We decided to make a transformation with our SST-genes even though the Nanodrop was a bit ambiguous.

We made PCR reaction on 16 different colonies from the FFT agar plate from the day before. For the first 8 tubes, we used primers standardized for the pJET1.2/BLUNT and the last 8 tubes we used the primers for extended FFT.
We ran a gel on the PCR products:
PCR 1-8, primer 3 and 4 and PCR 9-16, primer 354 and 355 (pJET1.2_for & pJET1.2_rev).
The gel gave inconclusive results for both sets of primers.

From the agar plate with FFT-ex clones, we took out 10 random colonies and put into liquid LB and put in the incubator at 37°C O.N.
The SST plate was also incubated O.N.