Dears, I made a colony PCR to verify my clones in that way: I picked up a colony to PCR mix: Buffer (10X) 3.0µL, DNTPs (2.5 mM each) 3.0µL, Taq (5 units/µL), Forward Primer (20 µM) Reverse Primer (20 µM) Water; TOTAL 30µL. Now I want to digest my PCR product by: BamHI an XhoI and the ligate insert to expression vector pET28b+. To save time and money I want to digest PCR mixture directly without any purification. I am not really sure that this way is correct. Do contaminations like bacteria cell components disturb restriction reaction? I know that they can disturb.... but I don't need 100% efficiency. So is there a posibility to obtain positive digestion and then ligation?
Please help me Marta _______________________________________________ Methods mailing list [email protected] http://www.bio.net/biomail/listinfo/methods
