3 replies to this topic

[Micro]

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  

[hanming86]

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.

[scolix]

I would purify the PCR product and then digest it.

[phage434]

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.