Phusion PCR: Difference between revisions
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*For difficult templates replace HF Buffer with GC Buffer and add DMSO or Betaine as required. | *For difficult templates replace HF Buffer with GC Buffer and add DMSO or Betaine as required. | ||
*Do not exceed 2 U polymerase/50 ul (0.04 U/ul), especially for amplicons > 5 kb. | |||
98 x 0:30, x (98 x 0:10, ___ x 0:20, 72 x ___), 72 x 7:00, 4 x Infinity | 98 x 0:30, x (98 x 0:10, ___ x 0:20, 72 x ___), 72 x 7:00, 4 x Infinity | ||
Primers and Tms | Primers and Tms | ||
Forward: | Forward: | ||
Reverse: | Reverse: | ||
Amplicons | |||
=TM Calculator for Phusion= | =TM Calculator for Phusion= | ||
[https://www.finnzymes.fi/tm_determination.html] | [https://www.finnzymes.fi/tm_determination.html] | ||
Latest revision as of 14:13, 22 September 2009
PCR Protocol for Phusion Polymerase
Reference: Adapted from New England BioLabs Phusion Hot Start High-Fidelity DNA Polymerase documentation.
| Components | Final Concentration | 1x Volume | Volume for Master |
|---|---|---|---|
| H2O | N/A | 33.1 ul | __ |
| 5x Phusion HF Buffer | 1x | 10 ul | __ |
| 25 mM DNTPs | 200 uM | 0.4 ul | __ |
| DMSO, Betaine, etc. | N/A | N/A | __ |
| Phusion Polymerase | 0.02 U/ul | 1 U = 0.5 ul | __ |
| Primers | 0.5 uM | 2.5 ul each = 5 ul | __ |
| Template DNA | N/A | 1 ul | __ |
- For difficult templates replace HF Buffer with GC Buffer and add DMSO or Betaine as required.
- Do not exceed 2 U polymerase/50 ul (0.04 U/ul), especially for amplicons > 5 kb.
98 x 0:30, x (98 x 0:10, ___ x 0:20, 72 x ___), 72 x 7:00, 4 x Infinity
Primers and Tms
Forward:
Reverse:
Amplicons
