Phusion
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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== | ||
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| + | 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== | ==Thermocycling conditions== | ||
| - | #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 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]] | ||
Current revision
Contents |
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.
