Phusion: Difference between revisions
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==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.'' | |||
{|{{Table}} | |||
http://www.neb.com/nebecomm/products/productF-530.asp | !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 | |||
| | |||
|- | |||
|H<sub>2</sub>O | |||
|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 than 20nt, else at the Tm. | |||
#*Note: Tm should be calculated with nearest neighbor method see: [http://www.finnzymes.fi/tm_determination.html Finnzyme Tm calculator] | |||
#Extend at 72C. | |||
==References== | |||
*[http://www.neb.com/nebecomm/products/productF-530.asp Purchase] | |||
*[http://www.neb.com/nebecomm/ManualFiles/manualF-540.pdf Manual] | |||
*[http://www.neb.com/nebecomm/products/productF-531.asp HF Master Mix product page] | |||
*[http://www.neb.com/nebecomm/products/productF-532.asp GC Master Mix product page] | |||
[[Category:Material]] [[Category:Enzyme]] [[Category:Polymerase]] [[Category:DNA]] | |||
Latest revision as of 16:12, 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 than 20nt, else at the Tm.
- Note: Tm should be calculated with nearest neighbor method see: Finnzyme Tm calculator
- Extend at 72C.
