Team:Groningen/Labwork/26 June 2013

Mirjam

Did a genomic DNA extraction of B. subtilis strain 168. Two tubes are obtained from this with a concentration of 66ng/µl and 67ng/µl 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.

Claudio and Inne

Further discussion about the available options to create a neat-attracted bacteria a discussed.