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Team:Groningen/Labwork/26 June 2013
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<br/>Extraction of the genomic DNA of ''B. subtilis'' strain 168. | <br/>Extraction of the genomic DNA of ''B. subtilis'' strain 168. | ||
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Run a 1.5% agarose gel for 13 minutes. Examination of the gel revealed that all expected products are there. The picture will follow. | Run a 1.5% agarose gel for 13 minutes. Examination of the gel revealed that all expected products are there. The picture will follow. | ||
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Revision as of 11:49, 24 July 2013
Mirjam
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.
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.
