Team:SDU-Denmark/labwork/Notebook/week2

Laboratory Notebook

Here you find the log book for the procedures carried out in the laboratory, starting from week 27.

06-07-2012:

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.).

10-07-2012:

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/ul. We did 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/ul.
With this result we decided to repeat the purification for the FFT-gene.
We tried to optimise the concentration of the FFT-gene from the gel and ended up with 13,8ng/ul determined by the NanoDrop.

We prepared another PCR for the SST-gene where we adjusted variables like raising the temperature to 63 degrees C for the annealing-step and adjusted the extension time.
PCR was run overnight.

11-07-2012:

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 (1% agarose gel) on the PCR product 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 gene to be. We also did a nanodrop analysis of our PCR products and got good results. A: 345,5 ng/uL (1A-4A) B: 436,2 ng/uL (1B-4B) (The gel extraction follows the guidelines of the Macherey-Nagel PCR clean-up Gel extraction User manual) The bands were cut out and the gel as the initial step of the gel extraction. For each 100mg gel, 200uL buffer NTI is added. We added X to a 770mg gel slice(4 bands) and X to a 650mg gel slice(4 bands). The samples were incubated at 50°C and vortexed every 2-3 mins until the slices were completely dissolved(about 10 minutes). A Nucleospin Gel clean-up column were placed into a collection tube(2mL) and the sample were added. The samples were centrifuged at 14.500rpm for 30 seconds. Flowthrough was discarded. 700uL NT3 buffer was added to the spin column, centrifuged at 14.500rpm, flowthrough was discarded. Previous washing step was repeated once. To dry the silica membrane of the spin column, the samples were centrifuged at 14.500rpm for 1 minute. the spin column was transferred to a new 1.5mL microcentrifuge tube. 30uL buffer NE was added and the samples were incubated at room temperature for 1 min, then centrifuged at 14.500rpm for 1 min. The solution in the bottom of the tube contained the extracted DNA. Subsequently the samples from the gel extraction were tested with Nanodrop 0,77g fragment 15,5ng/uL 0,65g fragment 35,7ng/uL The nanodrop test were very uncertain, we measured it several times and got different results, the above is an approximate that were made to determine the later dilution for PCR. The SST gene didn’t give any colonies, so we tried to do another ligation and transformation similar to the one yesterday, but with one exception (3 micro liter insert instead of 1, and the corresponding amount of water to match 10 micro liter in total). We made PCR reaction on 16 different colonies from the FFT agar plate. 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 left to right 1-8, marker, 9-16 From the agar plate with FFT-ex clones, we took out 10 random colonies and put into liquid LB and put in the incubator @ 37 degreeC O.N. The SST plate was also incubated O.N. The team tomorrow should: Contact Steffen, as he has a different approach to the way things should be done.