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
- - - - -

To clean or not to clean.....?


  • Please log in to reply
3 replies to this topic

#1 Micro

Micro

    Enthusiast

  • Active Members
  • PipPipPipPipPip
  • 60 posts
4
Neutral

Posted 25 February 2010 - 07:01 PM

I'm about to start a molecular cloning study on DNA from bacterial community. I will be screening my many, many clones for variations using RFLP. I have come across a papers that specifically state that they do the colony PCR on the clones, then add the RE and digest (no clean-up).

Does anyone out there have recommendations/opinions/experiences with skipping the PCR clean-up step and heading straight to RE digestion?

Cheers M :P

#2 hanming86

hanming86

    Veteran

  • Active Members
  • PipPipPipPipPipPipPipPipPipPip
  • 156 posts
2
Neutral

Posted 25 February 2010 - 07:30 PM

i would however recommend a purification step first.

This is to avoid having a suboptimal condition for the restriction digest. the salt present in the pcr reaction might affect the final salt concentration required for various restriction enzyme which eventually cause star activity or decrease or even inhibits the reaction.
Lab + Coffee + Music = Bliss

#3 scolix

scolix

    a student

  • Global Moderators
  • PipPipPipPipPipPipPipPipPipPip
  • 314 posts
4
Neutral

Posted 26 February 2010 - 08:36 AM

I would purify the PCR product and then digest it.

#4 phage434

phage434

    Veteran

  • Global Moderators
  • PipPipPipPipPipPipPipPipPipPip
  • 2,375 posts
227
Excellent

Posted 26 February 2010 - 06:33 PM

This depends entirely on what the purpose of the digestion is. Most REs will be active in PCR buffers. So if you want to do a pcr and then cut the product and run it on a gel as a diagnostic for picking out sequences that can be cut, then there is little reason to purify.

On the other hand, if you are preparing DNA for cloning after pcr, then it is a fatal mistake not to purify. The product of the RE will have (typically) a 5' overhang, which will be filled in by extending the 3' end of the RE cut site, destroying any ability to ligate with a compatible RE cut vector. You must purify the dNTPs and pcr enzyme away from the DNA prior to the cut.




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

©1999-2013 Protocol Online, All rights reserved.