Team:Groningen/28 June 2013

From 2013.igem.org

Mirjam
Examination of all PCR products made on 27-06-2013.

The silk product is run over a 0.8% agarose gel at 90V for 24 minutes.
The signal sequences are run over a 1.5% agarose gel at 90V for 14 minutes.
The gel of the signal sequence showed nice bands.
The gel of the silk again gave the lowest band as strongest. Only at 60 degrees Celsius a smear is seen, so this is the only one that also contains the higher bands.

Made a PCR for promoter Hyperspank LacI (PCR protocol as mentioned on 25-06-2013).
The following protocol is used:
98°C, 98°C, 50°C, 72°C, 72°C, 4°C
0:30 0:10 0:25 0:45 10:00 forever

Because of the failure of the same protocol with a gradient PCR of the silk. The PCR is done again only this time with the use of GC buffer instead of HF buffer. It is chosen to have two different annealing temperatures: 60 and 75 degrees Celsius
The following protocol is used:
98°C, 98°C, 60°C/75°C, 72°C, 72°C, 4°C
0:30 0:10 0:25 0:45 10:00 forever

A new 1.5% agarose solution is made and 1 ul/100 ml Serva is added.

Sander
Purified the obtained PCR product of the signal sequences.

Sander and Mirjam
Run a 1.5% agarose gel at 90V for 14 minutes of the purified PCR products
Run a 0.8% agarose gel at 90V for 30 minutes of the PCR product of the promoter and the silk(1).