phage434, on Jun 22 2009, 10:03 AM, said:
If you digest a PCR product without purifying the PCR enzymes and dNTPs, then the PCR enzymes will extend the 3' end of digested ends. This will destroy the site, and eliminate any possibility of ligating it with a vector.
I agree that some DNA polymerases can extent 3'dA, but there are some points:
1. To some percentage of PCR products (or digested products), this reaction is succeed (in the case of Taq polymerase, about 30%).
2. After PCR, DNA pol reaches or over its half-life time (I ran 30 cycles and 30 sec at 95 degree per cycle + initiation step at 95 degree for 5-10 minutes), so the active number of enzyme molecules and their activity reduce considerably.
3. The salt condition changes when I add RE buffer.
So, this activities just "destroy" a little amount of PCR products, but I gain the higher amount of DNA if I pass the purification step.
Actually, this works for me many times