Team:Groningen/26 June 2013

From 2013.igem.org

Extraction of the genomic DNA of B. subtilis strain 168.

Diluting the primers (to 100 mM and 10 mM)
Diluting the dNTPs to a concentration of 10 mM.

A PCR reaction mix is made containing the following compounds:
5x Buffer HF 1x
dNTPs 200 µM each
primer F 1 µM
primer R 1 µM
temp DNA 6.7 ng
phusion pol. 0.02 U/µl
MQ water is added to get the wanted volume.

Run a PCR for the four different signal sequences.
-FliZ
-MotB
-EstA
-LytB
The size will be around 100-150 bp.

The following protocol is used for the PCR of EstA, MotB and LytB:
98°C, 98°C, 50°C, 72°C, 72°C, 4°C
0:30 0:10 0:25 0:05 10:00 forever

The following protocol is used for the PCR of FliZ:
98°C, 98°C, 45°C, 72°C, 72°C, 4°C
0:30 0:10 0:25 0:04 10:00 forever

Run a 1.5% agarose gel for 13 minutes. Examination of the gel revealed that all expected products are there. The picture will follow.