I have been trying to cut pet22b(+) vector from novagen using Nde1 and BamH1 for my work, which requires ligation with Nde1 and BamH1.
I have tried the following way of cutting:
1) Cut with either enzyme first, gel purify, then cut with the other and purify again.
2) Double cut with both enzymes for a few hours and gel-purifying the product.
The above two gave me high background, so I got some high fidelity BamH1, which works in the same buffer as Nde1 and tried the following:
- Cutting with both Nde1 and BamH1(HF) overnight before gel-purifying.
- Cutting with both Nde1 and BamH1(HF) overnight before gel-purifying and taking the purified product for another round of cutting with both enzymes overnight before gel-purifying it again. This would mean double of double digestion and double gel-purification to get the cut vector.
Despite what I did above, I still got back high background during a test ligation without any inserts. With CIP treatment, the background was reduced significantly but this will not help me since I need the cut vectors for cloning, which will eventually be used for selection. Plus, all CIP does is to increase the chances of insert to vector ligation and reduce the changes of vector self ligation.
Thus, as a last resort, I ordered primers flanking the Nde1 and BamH1 RE regions on the vector and amplified the vector with Pfu before cutting it overnight with Nde1 and BamH1(HF) and gel-purifying it. What amazed me (in a not-so-happy way) was the presence of a high background! I do understand that self ligation can occur if the vector was not cut at all (since Pfu produces blunt ends). A single cut would not be able to generate any self ligation since one end would be blunt and the other cohesive. A double cut would not ligate too since the ends are incompatible. The enzymes cant be that inefficient, right? Especially since they are new enzymes and I have tried them out previously and saw double cut vectors for myself on the gel.
Now this is confusing me, is it THAT HARD to get a double cut vector with all the methods that I have tried out? Gel purifying ensures that I cut out and purify what I want but that is not happening. Some of the single-cut or uncut vector still gets purified along somehow. I do soak my gel tank with 0.1M HCl overnight to remove any traces of DNA to get the best possible purified band.
Now I am thinking of incubating my amplified vector with Taq 15mins after PCR to get A hangovers so that self ligation would be minimized. But nevertheless, could someone give me more suggestions?
I would appreciate any insights or help on this matter! I need double cut vectors!!! ARGH!!!
