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Team:Groningen/26 June 2013
From 2013.igem.org
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| - | Extraction of the genomic DNA ''B. subtilis''. | + | '''Mirjam''' |
| + | <br/>Extraction of the genomic DNA of ''B. subtilis'' strain 168. | ||
Diluting the primers (to 100 mM and 10 mM) | Diluting the primers (to 100 mM and 10 mM) | ||
<br/>Diluting the dNTPs to a concentration of 10 mM. | <br/>Diluting the dNTPs to a concentration of 10 mM. | ||
| + | |||
| + | A PCR reaction mix is made containing the following compounds: | ||
| + | <br/>5x Buffer HF 1x | ||
| + | <br/>dNTPs 200 µM each | ||
| + | <br/>primer F 1 µM | ||
| + | <br/>primer R 1 µM | ||
| + | <br/>temp DNA 6.7 ng | ||
| + | <br/>phusion pol. 0.02 U/µl | ||
| + | <br/>MQ water is added to get the wanted volume. | ||
| + | |||
Run a PCR for the four different signal sequences. | Run a PCR for the four different signal sequences. | ||
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<br/>-LytB | <br/>-LytB | ||
<br/>The size will be around 100-150 bp. | <br/>The size will be around 100-150 bp. | ||
| + | |||
The following protocol is used for the PCR of EstA, MotB and LytB: | 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 | + | <br/>98°C, 98°C, 50°C, 72°C, 72°C, 4°C |
| - | 0:30 0:10 0:25 0:05 10:00 forever | + | <br/>0:30 0:10 0:25 0:05 10:00 forever |
The following protocol is used for the PCR of FliZ: | The following protocol is used for the PCR of FliZ: | ||
| - | 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 |
| - | 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. Examination of the gel revealed that all expected products are there. The picture will follow. | ||
Latest revision as of 11:09, 28 June 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.
