Enzymetic digestion with ApaI and HincII - (Feb/02/2012 )
Dears, I amplied a new tag with primers having recognition sites for ApaI and HinCII, gel extracted the amplified fragments and then did the digestion with ApaI (incubation temperature at 25C) for three hours and kept it at 4C without heat inactivation. The next day I digested the same sample with HincII (ApaI and HincII both can work 100 % in NEB Buffer 4) at 37C for three hours and then heat inactivated. My question is since I didnt heat inactivate the sample after digestion with ApaI, was ApaI still active at 4C?.......basically I need to cut the extra fragments amplied with restriction enzymes and then to insert it into my vector.....
This should probably work, assuming you have sufficient 5' overhang in your PCR primers following the restriction sites. I would have done this with both enzymes in buffer 4, digesting first at 25 for a while (3 hours seems excessive, I'd probably get bored and use only 30 minutes) and then at 37, followed by a heat kill.
Normally, this is not a problem, but for some rare enzymes, it can be a problem by over digesting DNA.