Phusion

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(Thermocycling conditions)
 
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What is it?
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==Description==
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• a Fusion of a Pyrococcus-like enzyme (Pfu) with a double-strand DNA binding domain → increased processivity.
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Fusion of a Pyrococcus ([[Pfu]]) -like enzyme with a double-strand DNA binding domain → increased processivity.
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Stats about Phusion™ High-Fidelity DNA Polymerase:
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==Statistics about Phusion™ High-Fidelity DNA Polymerase==
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*10x processivity compared to Taq
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*50x fidelity compared to Taq
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*Creates blunt end
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*Error rate is 4.4 X 10-7 in Phusion HF buffer and 9.5 X 10-7 in GC buffer
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*Non-displacing
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• 10x processivity compared to Taq
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==Quick reference reaction mix==
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• 50x fidelity compared to Taq
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''See the manual for details and special usage conditions.''
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• creates blunt end
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• error rate is 4.4 X 10-7 in Phusion HF buffer and 9.5 X 10-7 in GC buffer
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• non-displacing
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Website:
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{|{{Table}}
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http://www.neb.com/nebecomm/products/productF-530.asp
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!Component
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!Volume for 50μl reaction
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!Final concentration
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|-
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|5x Phusion HF Buffer
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|10μl
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|1x
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|-
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|10mM dNTPs
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|1μl
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|200μM each
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|-
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|primer A
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|x μl
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|0.5μM
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|-
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|primer B
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|x μl
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|0.5μM
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|-
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|template DNA
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|xμl
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|
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|-
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|H<sub>2</sub>O
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|add to 50&mu;l
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|
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|-
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|Phusion DNA polymerase (2U/&mu;)
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|0.5&mu;l
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|0.02 U/pl
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|}
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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.
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==Thermocycling conditions==
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#15-30 s/kb extension time.
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#98C for denaturation.
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#Anneal at 3C above the lowest Tm if the primers are longer than 20nt, else at the Tm.
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#*Note: Tm should be calculated with nearest neighbor method see: [http://www.finnzymes.fi/tm_determination.html Finnzyme Tm calculator]
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#Extend at 72C.
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==References==
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*[http://www.neb.com/nebecomm/products/productF-530.asp Purchase]
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*[http://www.neb.com/nebecomm/ManualFiles/manualF-540.pdf Manual]
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*[http://www.neb.com/nebecomm/products/productF-531.asp HF Master Mix product page]
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*[http://www.neb.com/nebecomm/products/productF-532.asp GC Master Mix product page]
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[[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

  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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