Cloning problem using pET32a vector - (Jan/24/2012 )

Hello, I am new to the forums, but have found a lot of helpful information here. I am trying to insert a 2kb fragment into a 5.9kb pET32a vector. The insert (2kb) has a blunt end (from the PCR amplification) and an EcoRI site at the end. The pET32a vector was digested with NdeI and blunted with pfu fusion polymerase and then digested with EcoRI. My problem is I get no colonies after ligation. Here is what I have tried.

Transformation of uncut pET32a gives a lawn of bacteria on LB + Amp agar (vector is OK)
2hr, 4hr, and overnight NdeI and EcoRI digests give the same results - no colonies
After each digest, and pfu fill-in both vector and insert are purified by Qiaquick gel extraction
I have tried a 1:3 and 1:8 vector to insert ratio
I have tried both NEB Quick ligase and Promega T4 DNA ligase

One tricky part is that it is not possible to tell if the ecoRI digestion was successful by gel visualization since very few bp are lost in the digestion. Also, I lose much of my vector after pfu fill-in and by the end of all of the digests and purification I am only left with about 40-50 ng of vector.

Any advise on this problem would be greatly appreciated. I am leaning towards the problem being not enough vector and I am not sure on how to go about increasing my yield. Also, I get a bit of a smear/double band after I digest pET32a with NdeI even overnight which might indicate incomplete digestion.

You can tell if your EcoRI digest is working by ligating the fragment (either the insert or the vector) with itself, and looking for the double length linear result. If you use normal T4 ligase buffer and heat kill the reaction, you will get little blunt ligation.

Have you considered using PCR to amplify your vector, adding whatever enzyme sites you want to the (now linear) cloning site?

I would check the UV exposure of my gels, which can often dramatically reduce transformation efficiency.

Thanks for the tip and I do tend to leave the gel exposed longer than I should. I havent considered amplifying the vector by PCR but instead I might design primers to amplify the insert with both a EcoRI and XbaI restriction site to do a sticky end ligation with the double digested vector. Do you feel that this will increase the likliehood of success by eliminating 2 gel purification steps and a blunting step with the vector?

Thanks

I would definitely choose a redesign of your primers instead of trying to make a mixed blunt/overhang ligation work.