Team:Groningen/protocols/Colony PCR
From 2013.igem.org
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Revision as of 16:05, 27 September 2013
Colony PCR
Materials:
- PCR tubes
- MQ water
- DMSO 5%
- Phusion buffer
- DNTPs
- F- and R-primer
- Phusion Polymerase
- DNA template
PCR reaction mixture:
Component | 20 µl | Final concentration |
---|---|---|
MQ water | up to 20 µl | |
5x Phusion Buffer | 2 µl | 1x |
DMSO 5% | 1.5 µl | |
10 mM dNTPs | 1 µl | 200 µM |
primer F | 1 µl | 0.5 µM |
primer R | 1 µl | 0.5 µM |
template DNA | 1 µl | ∼1 ng |
Phusion Polymerase 2U/µl |
0.3 µl | 0.01U/µl |
Cycling instructions:
Temperature | Time | Number of Cycles |
---|---|---|
98°C | 10 min | 1 |
98°C | 10 sec | 34 |
Tm-5°C | 25 sec | |
72°C | 30 sec/kb | |
72°C | 10 min | 1 |
4°C | ∞ | 1 |