Team:Groningen/Labwork/1 July 2013

Mirjam

Made a PCR reaction for the promoter. Using the same PCR protocol as 28-06-2013. This time in the PCR mix the template DNA is the PCR product of friday and instead of 1 ul, 3 ul is used. To get a higher concentration of product. Also a new PCR reaction is made for the silk(1). Because it is not known what the best PCR method is, this time the PCR is done in duplo with an annealing of 45 and 55 degrees Celsius (step 2-4 is repeated 34 times). For both a combination is made with and without DMSO. This all to optimize the protocol.
1 2 3 4 5 6
98 98 45/55 72 72 4
2:00 0:10 0:25 0:27 10:00 endless Unfortunately at this stage, the power supply in the lab is shut down. Therefore there might be a difference when the reactions are run again. Fortunately the PCR starts at the point where it is shut down before.

Mirjam and Sander

The PCR reactions are run over a 0.8% agarose gel for 22 minutes at 100V. Gel examination revealed that the promoter is not present. The silk is getting better by improving the protocol. Still no band is seen at 900 bp. But the bands at the lower ranges are appearing. There is no clear difference between the product with DMSO and without. A new PCR is made to obtain the promoter. This time 1 ul of genomic DNA is used and the PCR is performed in duplo.
1 2 3 4 5 6
98 98 55 72 72 4
2:00 0:10 0:25 0:45 10:00 forever For the silk it is decided to also try a PCR with an annealing temperature of 40 degrees Celsius. As it turned out that the lower the temperature the clearer the bands are. This time it is chosen to do a very long denaturation time.
1 2 3 4 5 6
98 98 40/45 72 72 4
20:00 0:30 0:25 0:27 10:00 forever

Sander

The PCR reactions are run over a 0.8% agarose gel for 22 minutes at 100V. Gel examination revealed that the promoter is present in one of the PCR runs. The silk is has no improvement. Similar results as before. There is no clear difference between the product with DMSO and without.
Purification of promotor (vial 1) planned for tomorrow