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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).

The gels are examined. This revealed an improvement of the PCR product of silk(1), but the PCR product of the promoter did not show the correct bands.
Also the purified PCR product of the signal sequences are examined which revealed that the product is present. That is why the concentration is measured. the following results are obtained:
LytB 33,9 ng/ul
MotB 49,5 ng/ul
FliZ 52,2 ng/ul
EstA 42,1 ng/ul

Because the PCR of the silk did show improvement, the GC buffer will be kept on. A new reaction is made with the addition of DMSO. The protocol of the PCR reaction is kept the same. Also new reaction of the promoter is made. The following protocol is used:
98°C, 98°C, 55°C, 72°C, 72°C, 4°C
0:30 0:10 0:25 0:45 10:00 forever

A 0.8% agarose gel is made and run for 22 minutes at 90V.
Gel examination revealed that the change of getting a piece of 900 bp of silk is higher when the annealing temperature is lower and the denaturation time is longer. The promoter LacI is present although in a very low concentration.