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Team:Groningen/protocols/Colony PCR
From 2013.igem.org
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Revision as of 11:24, 28 July 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 | 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 |
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