I am attempting to ligate a 2.2 kb fragment into a 2.6 kb vector. The vector is not commercially available and was made by our lab. It is a low copy number vector. I have tried the ligation several different ways. I use T4 DNA ligase. I've tried all ligations with different amounts of total DNA, as low as about 20 total and as much as about 120 total. I use homemade DH5alpha competent cells, 10 microL ligation in 90 microL competent cells.
1. PCR amplifying my fragment with Herculase, cutting it with BamHI and SalI, and ligating it into my vector cut with the same enzymes. I have sequenced my PCR frag and know it is what I am expecting. I can tell my fragment is cut with both enzymes, becuase there is about 50 bp on each side of the fragment eliminated with the cuts (enough to see a size difference on a gel). I use a 1:3 and a 1:5 ratio, and a control with no insert. I have good ratios on my plates (almost no colonies on control and about 50 total on both the other plates combined). I don't have a PCR screen that works, so I miniprep and cut all of them with the same enzymes I used in the ligation - BamHi and SalI. Hence a positive should have a 2.6 kb band (vector) and a 2.2 kb band (insert). In about 3 in every 50ish colonies, I get something that looks like a positive, only it's not. The "vector" band is about 300 bp too large.
2. I have also PCR amplified by fragment in the same manner as above, only using a primer with an Acc65I cutsite (and buffer nucleotides so the cutsite is not right at the end). I cut the fragment with Acc65I and leave the other end blunt, and ligate into my vector in the same manner as above, but the vector is cut with EcoRV and Acc65I. In this case I have no way of telling for sure that my fragment is cut. In this case, my ratios are not good, but I have a lot more colonies (in the hundreds on ligation plates and noticibly less but still a lot on the control). I screen the colonies and see something very similiar to above.
3. I have also cut my fragment directly out of it's plasmid using BamHI and XhoI, which has a compatible end with SalI. I gel extract the fragment with the Qiagen kit. When I do the ligation, I get no colonies on any of the plates.
I know this suggests I am getting something nonspecfic from my PCR that is ligating into my vector and being taken up my my competent cells. I am planning to gel extract my PCR frag and try the ligation, and I actually have but it hasn't been conclusive (contamination and other problems). Does anyone have any clue why this isn't working? I know it's difficult to ligate large inserts into small vectors, but I have no clue where to go from here.