Go Back   Science Forums Biology Forum Molecular Biology Forum Physics Chemistry Forum > Molecular Research Topics Forum > PCR - Polymerase Chain Reaction Forum
Register Search Today's Posts Mark Forums Read

PCR - Polymerase Chain Reaction Forum PCR - Polymerase Chain Reaction Forum. Discuss and ask questions about PCR troubleshooting, PCR protocols and methods, PCR products, and PCR theory.


Replacing Taq with Pfu in PCR reaction

Replacing Taq with Pfu in PCR reaction - PCR - Polymerase Chain Reaction Forum

Replacing Taq with Pfu in PCR reaction - PCR - Polymerase Chain Reaction Forum. Discuss and ask questions about PCR troubleshooting, PCR protocols and methods, PCR products, and PCR theory.


Reply
 
LinkBack Thread Tools Display Modes
  #1  
Old 03-18-2011, 06:20 PM
Pipette Filler
Points: 4, Level: 1 Points: 4, Level: 1 Points: 4, Level: 1
Activity: 0% Activity: 0% Activity: 0%
 
Join Date: Mar 2011
Posts: 1
Thanks: 0
Thanked 0 Times in 0 Posts
Default Replacing Taq with Pfu in PCR reaction



Hi guys. I was wondering if anyone have any experience replacing Taq with Pfu with PCR reactions? Under what circumstances would they work and under what circumstances would this not work?
Reply With Quote
  #2  
Old 03-26-2011, 10:34 AM
Pipette Filler
Points: 1,111, Level: 19 Points: 1,111, Level: 19 Points: 1,111, Level: 19
Activity: 0% Activity: 0% Activity: 0%
 
Join Date: Oct 2010
Posts: 7
Thanks: 0
Thanked 1 Time in 1 Post
Default Re: Replacing Taq with Pfu in PCR reaction

i did use pfu( Fermentas) to amplify a band after i optimized the condition using taq. but the result is not good. multiple bands were obtained although only single band was produced by taq. pfu not works well for my case, i think proble due to the pfu.because i try a few enzyme to amplify the same product(2.5kb). pfu took me 4-5 hours, and the yield very low, not suitable for cloning.
now i m looking for other enzyme with high fidelity and proofreading activity to amplify the product. hope someone can tell me what enzyme is good for my case, thank you.
Reply With Quote
  #3  
Old 03-28-2011, 05:49 AM
Graduate Student
Points: 3,129, Level: 36 Points: 3,129, Level: 36 Points: 3,129, Level: 36
Activity: 0% Activity: 0% Activity: 0%
 
Join Date: Aug 2008
Posts: 108
Thanks: 4
Thanked 48 Times in 42 Posts
Default Re: Replacing Taq with Pfu in PCR reaction

It depends. Pfu is more selective and difficult work with. I use regular Taq to do a quick screening whether the PCR reaction works (template/primer design etc) before I switch to Pfu.
Reply With Quote
  #4  
Old 03-28-2011, 08:21 AM
Pipette Filler
Points: 938, Level: 17 Points: 938, Level: 17 Points: 938, Level: 17
Activity: 0% Activity: 0% Activity: 0%
 
Join Date: Mar 2011
Location: Damascus-Syria
Posts: 7
Thanks: 0
Thanked 0 Times in 0 Posts
Default Re: Replacing Taq with Pfu in PCR reaction

hi, I'm using Pfu just in case of isolation , cloning and sequencing to make sure from sequence fidelity .. but PCR reaction must be optimised (DNA tempalt and primers..etc) and in some cases may be u have to use PCR enhancer if there is no product or low yield... in PCR programme the extension tm depends on the size of the amplified fragment.. 1 min per Kb decreasing cycles number to minimize non-specific products
Reply With Quote
  #5  
Old 05-10-2011, 06:23 AM
Pipette Filler
Points: 65, Level: 1 Points: 65, Level: 1 Points: 65, Level: 1
Activity: 0% Activity: 0% Activity: 0%
 
Join Date: May 2011
Posts: 2
Thanks: 0
Thanked 0 Times in 0 Posts
Default Re: Replacing Taq with Pfu in PCR reaction

Hi i have used pfu for amplifying the high GC rich regions and mitochondrial genome. It worked well for me , initially i did get a few nonspecific bands but later they diappeared.
Reply With Quote
Reply

Tags
pcr , pfu , reaction , replacing , taq


Thread Tools
Display Modes

Posting Rules
You may not post new threads
You may not post replies
You may not post attachments
You may not edit your posts

BB code is On
Smilies are On
[IMG] code is On
HTML code is Off
Trackbacks are On
Pingbacks are On
Refbacks are On

Forum Jump

Similar Threads
Thread Thread Starter Forum Replies Last Post
What does order of a reaction tell us? SNUMBER6 Chemistry Forum 29 06-25-2013 04:21 PM
Repetitive reaction for random priming? Ryohichi Molecular Biology Techniques 0 06-07-2009 02:41 AM
Reaction mechanism and reaction products zhangtao@gmail.com Organic Chemistry Forum 0 12-19-2005 09:07 AM
combustion/ionisation physics Peter Lowrie Physics Forum 3 06-20-2005 01:56 PM
CIP reaction not working... Raghuvir Viswanatha Protocols and Methods Forum 4 10-22-2003 05:09 PM


All times are GMT. The time now is 06:22 PM.


Powered by vBulletin® Version 3.8.4
Copyright ©2000 - 2014, Jelsoft Enterprises Ltd.
Copyright 2005 - 2012 Molecular Station | All Rights Reserved
Page generated in 0.13449 seconds with 16 queries