Hi everyone.
I am working on making RNA probes for in situ hybridization, and am at the moment about to transform some competent cells. However, I just realized that the concentration of the vector that I am using (Zero-Blunt TOPO vector) is only 10 ng/ul, where I can use up to 1000 ng for the ligation in the protocol that I am following. I am not following the TOPO protocol because I want to do restriction digestion, where the TOPO vector protocol assumes that you are using the blunt ends. I tried doing restriction digestion on the TOPO vector to get the ends sticky and run it on gel, but I got no band up, supposedly because I only used 20 ng. The problem is that I guess I will have to use pretty big volumes to get a visible band on the gel (which I will need to cut out afterwards), but the amount that I have of the TOPO vector is not very large. Also, the TOPO vector I am using is already blunt-ended, so I was wondering if it is possible to first make it circular and than do some transformation on competent cells just to get more vector?
The T4 DNA ligase should ideally work on blunt ends as well, but I have never done it. However, you can probably give it a try. Maybe try using around 10ng of your linearized vector in a 5ul ligation mix and then transform the whole thing. I hope it helps.
- Badges
- Science topic
- Similar topics
- Analytical Chemistry
- Extraction