I'm trying to construct a small insert metagenomic library and I can't seem to get any colonies - ligations.

Question

After purifying my DNA, (with the bioflux kit or glass milk) I had lost a lot of DNA. I tried to ligate into pZErO-2 as I'm doing cohesive end ligations (16oC ;16H, 4oC 16H, RT 1H). I even change the ligase and it's buffer. I checked my competent cells and they are fine, even my test insert is not working!

Please help, I don't know what else to do!

Answer 1

1) Glass milk purification can be problematic when you wish to use the DNA for ligations. I used the Bio101 kit for months before I realized this. Change your purification method: I suggest Qiagen spin columns.

2) You can use alkaline phosphatise to treat the target vector. This will remove the 5’ phosphates, preventing the vector from closing on itself. Most people don’t do this anymore, but it can help.

3) What is the source of your insert? If you are generating it by PCR, or can generate it by PCR, then try TA cloning (kits are available, I liked Promega's the best, but go with what works for you). This is especially useful if you have a blunt end fragment as blunt ligations suck. If memory serves pZero is not an expression vector, so frame should not be an issue. If frame is an issue, you may have to design PCR primers that compensate for this.

4) Make sure you calculate your ratio of insert to vector correctly. Check here for the calculation: http://openwetware.org/wiki/DNA_Ligation...

5) Make sure there is NO ethidum bromide present in either the ligation reaction or the transformation.