"
Team:Groningen/26 June 2013
From 2013.igem.org
| Line 29: | Line 29: | ||
<br/>98°C, 98°C, 45°C, 72°C, 72°C, 4°C | <br/>98°C, 98°C, 45°C, 72°C, 72°C, 4°C | ||
<br/>0:30 0:10 0:25 0:04 10:00 forever | <br/>0:30 0:10 0:25 0:04 10:00 forever | ||
| + | |||
| + | Run a 1.5% agarose gel for 13 minutes. | ||
Revision as of 14:37, 26 June 2013
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.
