Jump to content

  • Log in with Facebook Log in with Twitter Log in with Windows Live Log In with Google      Sign In   
  • Create Account

Submit your paper to J Biol Methods today!
Photo
- - - - -

PCR to get 10kbp product


  • Please log in to reply
4 replies to this topic

#1 DrLeo

DrLeo

    Enthusiast

  • Active Members
  • PipPipPipPipPip
  • 40 posts
3
Neutral

Posted 17 June 2013 - 08:48 PM

Hi all,
I have to get 10kbp PCR product.
I have 2 primers, which have Tm of 58oC (F) and 60oC ®. I intend to use Pfu Taq pol to get the high accuracy.
What should I do to get success?
Thank you so much for your suggestion!

#2 jerryshelly1

jerryshelly1

    Veteran

  • Global Moderators
  • PipPipPipPipPipPipPipPipPipPip
  • 328 posts
33
Excellent

Posted 18 June 2013 - 05:58 AM

I have used Pfu to get a 7.5Kb product, no problem. I have included a link to the Pfu polymerase PCR reaction setup. With longer PCR products you need to be aware of the GC content of your gene of interest. For example, when I was cloning my gene, my product would not amplify without the presence of GC enhancer (my gene had high GC content ~60%, with alot of GC repeats). I would follow the included protocol and add the highest amount of dNTP's (1uM). If you have a gradient PCR machine, I would recommend trying a couple of different reaction conditions. Maybe one with higher dNTPs incase they are exhausted in your reaction, one with GC enhancer, possibly a couple of different annealing temps. You shouldn't have any problems. Good luck.


http://www.thermosci...tion-set-up.pdf

#3 DrLeo

DrLeo

    Enthusiast

  • Active Members
  • PipPipPipPipPip
  • 40 posts
3
Neutral

Posted 27 June 2013 - 02:26 AM

Thanks Jerryshelly,
As you mentioned, the Elongation step you used a period of 15minutes, is it?
And the GC enhancer you mentioned is located in the target gene or somewhere else???
Thanks again

#4 phage434

phage434

    Veteran

  • Global Moderators
  • PipPipPipPipPipPipPipPipPipPip
  • 2,499 posts
252
Excellent

Posted 27 June 2013 - 05:39 AM

I would recommend Phusion or Q5 polymerase, and an extension time of only 3 minutes. Make sure you follow their cycling temperature recommendations.

#5 DrLeo

DrLeo

    Enthusiast

  • Active Members
  • PipPipPipPipPip
  • 40 posts
3
Neutral

Posted 27 June 2013 - 06:08 AM

Dear Phage434,
Thank you very much for your suggestion. It really makes sense to me.
However, have you ever tried with Q5 pol for amplifying large fragment, say > 10kbp?
Thank you again




Home - About - Terms of Service - Privacy - Contact Us

©1999-2013 Protocol Online, All rights reserved.