Phusion

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(Quick reference reaction mix)
(Thermocycling conditions)
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==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 Tm.
 +
#Extend at 72C.
==References==
==References==

Revision as of 11:01, 19 July 2007

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

Thermocycling conditions

  1. 15-30 s/kb extension time.
  2. 98C for denaturation.
  3. Anneal at 3C above the Tm.
  4. Extend at 72C.

References

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