Muratore:Protocols/PCR/Quikchange
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Tube | sterile H2O | Pfu Buffer | Template | For primer | Rev primer | dNTPs | Pfu Turbo | wax |
---|---|---|---|---|---|---|---|---|
(10X) | (10 μg/mL) | (12.5 ng/μL) | (12.5 ng/μL) | (10 mM ea) | (2.5 U/μL) | |||
Experimental | 12 μL | 10 μL | 10 μL | 10 μL | 5 μL | 2 μL | 1 μL | 50 μL |
(-) Control | 13 μL | 10 μL | 10 μL | 10 μL | 5 μL | 2 μL | 0 μL | 50 μL |
- Add each from left to right. Mix and spin before adding Pfu Turbo. Gentle mixing and spin after Pfu Turbo. Don't mix in wax.
- 30" @ 95°C
- 30" @ 95°C
- 1' @ 55°C
- 8.5' @ 68°C
- repeat steps 2-4 x 15 (for one base change) or 17 (for more than one base change) more times
- 10' @ 68°C