Is T4 DNA Polymerase so evil? Blunt End Cloning - (Feb/16/2011 )

Hey there

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.

Greetings
Lucas

Hi Lucas,
I think you should look into:
Fast DNA End Repair Kit K0771 (Fermentas) http://www.fermentas.com/en/products/all/molecular-cloning/kits/k077-fast-dna-end-repair
This kit is more suitable to do the trick.

Also, you did mentioned that you do add dNTPs and T4 polymerase at the end of the restriction digestion. I think you should try to purify your digested fragments before you add in the said both item.

Just my 2 cents.

Thank for the reply.

I will try again with the additional cleanup step.

I am not sure about the kit you suggested. I mean its basically just a mixture of Klenow, T4 DNA Polymerase and T4 Polynucleotide Kinase. T4 PNK I don't think I need, DNA was prepared by RE digest, and is therefore phosphorylated. Klenow and T4 DNA Pol should both do the trick..

The reaction has to work with T4 DNA Pol alone, so I am not very keen on purchasing a kit at twice the price..

Why cant I find the edit button?

Anyhow, I think i got it sorted, still have to screen the colonies, but i had 15 vs 2 colonies on control plate. Additional Purification before blunting and ligating for 24h at 4 degrees did the trick.