Phusion: Difference between revisions
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==Description== | ==Description== | ||
Fusion of a Pyrococcus (Pfu) -like enzyme with a double-strand DNA binding domain → increased processivity. | Fusion of a Pyrococcus ([[Pfu]]) -like enzyme with a double-strand DNA binding domain → increased processivity. | ||
==Statistics about Phusion™ High-Fidelity DNA Polymerase== | ==Statistics about Phusion™ High-Fidelity DNA Polymerase== | ||
Line 46: | Line 46: | ||
|0.02 U/pl | |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== | ==References== | ||
*[http://www.neb.com/nebecomm/products/productF-530.asp Purchase] | *[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/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]] | [[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.