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
- - - - -

pooling different pcr cycles?


  • Please log in to reply
1 reply to this topic

#1 claritylight

claritylight

    Veteran

  • Active Members
  • PipPipPipPipPipPipPipPipPipPip
  • 104 posts
0
Neutral

Posted 12 December 2010 - 11:49 AM

If one is running low on template and needs more volume for PCR cleanup, can PCR products from different cycles be pooled together for example pool an 5 cycle PCR reaction with a 10 cycle PCR reaction? Say the PCR reactions were made from the same mastermix, only different cycles were done and both cycles gave very good product.

Would pooling the different cycles increase the final concentration of the pool or dilute it? And what would be the drawbacks from doing such a thing, besides risking more primer dimers in the final pool (which can be probably be cleaned up anyway).

#2 perneseblue

perneseblue

    Unlimited ligation works!

  • Global Moderators
  • PipPipPipPipPipPipPipPipPipPip
  • 578 posts
17
Good

Posted 12 December 2010 - 02:40 PM

well it depends if the plateau phase has been achieve after that many cycles.
If both PCR reaction mixes have reached the plateau phase than mixing the two could not change the concentration. Since the concentration of PCR product would be identical.

However if the plateau phase has not been achieved, then the reaction mix with the fewer number of cycles would logically has less product, and thus a lower concentration. Pooling the two would increase the amount of PCR product present but decrease the concentration.

I would add 5 cycle even 10 cycles is rather few. I use 25 cycle to amplify my PCR product.

Aside from not being as efficient as possible, I don't see a problem pooling the two.
May your PCR products be long, your protocols short and your boss on holiday




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

©1999-2013 Protocol Online, All rights reserved.