I have problems with BLunt end cloning. I am using T4 DNA Polymerase (Fermentas) to generate Blunt Ends both on vector and on insert.
I tried according to manufacturers protocol (which also includes the use of PEG4000), but even vector alone + ligase resulted in no colonies. So there is a problem with Blunting Reaction.
I added dNTPs and T4 DNA Polymerase at the end of Restriction digestion, because Fermentas states that the Polymerase is active in RE Buffers. Could this be a source of the problem?
Afterwards I gel purify with QIAGEN QIAEX II Gel extraction kit.
Could the problem also be due to vector destroyed by T4 DNA Polymerase? Would it make sense to try different incubation times (e.g. 2,5,8 minutes) and different enzyme concs?
After all, I am quite fed up with cloning. I want to assemble like 12 lentiviral vectors and got some of sticky end (from excised insert as well as PCR Product with RE sites) to work, but the efficiency was always very poor (ten colonies or so). So Im still thinking that there is a general problem. But I am paying attention to all of the popular suggestions (3:1 ratio, use clean DNA, Ligate at 4°C overnight).
Has anyone experience in using Fermentas products for Cloning? All my enzymes are from them (RE, FastAP to dephosphorylate, Ligase, T4 DNA Polymerase .....). The ligase works ok when assayed on a plasmid cut with a single RE. I am somehow disappointed that the manufacturers instructions do not seem to do the trick (Ok, maybe I am being naive here ) Unfortunately I have no supervision that could help me with cloning in any way, so any suggestions would be greatly appreciated.
For me, cloning really remains voodoo to some extent. I know how it should work, but it simply doesn't. There are so many different protocols and suggestions that I have no idea what I should be trying.. Quite frustrating.
Edited by loucash, 16 February 2011 - 07:11 AM.