To clean or not to clean.....?
Posted 25 February 2010 - 07:01 PM
Does anyone out there have recommendations/opinions/experiences with skipping the PCR clean-up step and heading straight to RE digestion?
Posted 25 February 2010 - 07:30 PM
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.
Posted 26 February 2010 - 06:33 PM
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.