Phusion

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(Thermocycling conditions)
Current revision (19:12, 13 February 2013) (view source)
(Thermocycling conditions)
 
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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 lowest Tm if the primers are longer then 20nt, esle at 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: [https://www.finnzymes.fi/tm_determination.html Finnzyme Tm calculator]
+
#*Note: Tm should be calculated with nearest neighbor method see: [http://www.finnzymes.fi/tm_determination.html Finnzyme Tm calculator]
#Extend at 72C.
#Extend at 72C.

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

  1. 15-30 s/kb extension time.
  2. 98C for denaturation.
  3. Anneal at 3C above the lowest Tm if the primers are longer than 20nt, else at the Tm.
  4. Extend at 72C.

References

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