Phusion: Difference between revisions
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(→Thermocycling conditions: comment on Tm calculation method) |
Jarle Pahr (talk | contribs) |
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#15-30 s/kb extension time. | #15-30 s/kb extension time. | ||
#98C for denaturation. | #98C for denaturation. | ||
#Anneal at 3C above the Tm. | #Anneal at 3C above the lowest Tm if the primers are longer then 20nt, esle at the Tm. | ||
#*Note: Tm should be calculated with nearest neighbor method see: [https://www.finnzymes.fi/tm_determination.html Finnzyme Tm calculator] | #*Note: Tm should be calculated with nearest neighbor method see: [https://www.finnzymes.fi/tm_determination.html Finnzyme Tm calculator] | ||
#Extend at 72C. | #Extend at 72C. |
Revision as of 09:01, 13 February 2013
Description
Fusion of a Pyrococcus (Pfu) -like enzyme with a double-strand DNA binding domain → increased processivity.
Statistics about Phusion™ High-Fidelity DNA Polymerase
- 10x processivity compared to Taq
- 50x fidelity compared to Taq
- Creates blunt end
- Error rate is 4.4 X 10-7 in Phusion HF buffer and 9.5 X 10-7 in GC buffer
- Non-displacing
Quick reference reaction mix
See the manual for details and special usage conditions.
Component | Volume for 50μl reaction | Final concentration |
---|---|---|
5x Phusion HF Buffer | 10μl | 1x |
10mM dNTPs | 1μl | 200μM each |
primer A | x μl | 0.5μM |
primer B | x μl | 0.5μM |
template DNA | xμl | |
H2O | add to 50μl | |
Phusion DNA polymerase (2U/μ) | 0.5μl | 0.02 U/pl |
A 2x supermix is now available containing either HF buffer or GC buffer, dNTPs, and Phusion polymerase. See references section below for links.
Thermocycling conditions
- 15-30 s/kb extension time.
- 98C for denaturation.
- Anneal at 3C above the lowest Tm if the primers are longer then 20nt, esle at the Tm.
- Note: Tm should be calculated with nearest neighbor method see: Finnzyme Tm calculator
- Extend at 72C.