It's my understanding that due to the 3'-5' exonuclease activity of the Phusion polymerase, the PCR-product will NOT have any A-overhangs. Meaning the PCR-product will have blunt ends.
So in order to follow the experiment up with TOPO-cloning. I need to create A-overhangs. Has anyone done this setup with Phusion-TOPO and can give some feedback/advices?
1) Phusion PCR (already done and stored in -20C for 4weeks)
2) Purify the PCR-product from remaining Phusion polymerase/dNTPs/primers/etc
3) Add 0.7 - 1 unit Taq polymerase (not necessary to change buffer)
4) Incubate 72C, 10min
5) Use directly in the TOPO cloning system
All steps performed on ice (except incubation)
Should I be concerned that the PCR-product (that is ALREADY made, but has no A-overhangs) have been in the -20C for close to 4weeks? Would that affect the results?
My supervisor doesn't want me to make any fresh PCR-products. Instead I use what I got. And also the simplest would be to generate a new PCR-product using the Taq-polymerase (hence no need to artificially create the overhangs alter), but that would require to buy the kit and I guess the supervisor once again wants to use what he already has in the lab.