Hi everyone,
I want to carry out some DNA FISH experiments on different genomic regions ranging from 12 to 19 Kb in length. My strategy would be to first amplify them by PCR and use the purified product as the nick translation substrate. The reasons why I am not directly labelling the probe by PCR with modified nucleotides are 1) I already have a nick translation kit and 2) nick translation produces shorter probes which work much better in my conditions.
Question: can nick translation be successfully performed on linear PCR products? I've already successfully used it before but on fosmids.
Many thanks!
Best,
Andrea
Hello, Nick translation will only generate small fragments of < 325 bp which may not be suitable for getting a good signal. Better to label the probe by incorporation of fluorescent dNTPs. You will get a better probe of defined size and probably greater fluorescence. But if you want to try Nick translation go ahead !
Hello, Nick translation will only generate small fragments of < 325 bp which may not be suitable for getting a good signal. Better to label the probe by incorporation of fluorescent dNTPs. You will get a better probe of defined size and probably greater fluorescence. But if you want to try Nick translation go ahead !
Karen A. Darbinyan many thanks for your reply. As I stated in my question, I prefer to use nick translation to produce probes because, first I was told by a person with extensive DNA FISH experience that it generally work better than directly labelling by PCR, and secondly because shorter probes are easier to get into the sample instead of large ones, especially in my sample.
- Badges
- Science method