PCR
Materials:
- PCR tubes
- F- and R-primer
- DNTPs
- Phusion buffer
- H2O
- Phusion Polymerase
- DNA template
Steps:
| Component |
50μl reaction |
Final concentration |
| H2O |
up to 50μl |
|
| 5x Phusion Buffer |
10 |
1x |
| 10mM dNTPs |
1 |
200μM |
| F-primer |
1 |
0.2μM |
| R-primer |
1 |
0.2μM |
| Template DNA |
1μl |
~1ng |
| Phusion Pol. |
0.5μl |
0.02U/μl |
Cycling instructions:
| Temperature |
Time |
Number of cycles |
| 98°C |
30sec |
1 |
| 98°C |
10sec |
30 |
| primer annealing temperature |
30sec |
30 |
| 72°C |
30sec/kb |
30 |
| 72°C |
10min |
|
| 4°C |
∞ |
1 |
Reference: http://www.thermoscientificbio.com/uploadedFiles/Resources/f-530s-product-information.pdf
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