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