I want to use Sanger sequencing to sequence my PCR product. I was having trouble with a nonspecific PCR product (A) that was coming up, so I ran my reaction on a temperature gradient. The nonspecific product disappeared at higher temperatures like I wanted, but a couple of new bands emerged (B). What do they mean? How should I respond?
If you have enough PCR Product and you can seperate the bands sufficiently by electrophoresis you can simply cut out the correct band and clean this up and use the purified PCR product for sequencing . If you only have relative small amounts of the PCR product you can do the same and try to run the PCR again on the purified PCR product to improve your DNA yield.
You can also try DMSO in your PCR reaction - this can improve the specificity of the reaction ( e.g. 1,25 µl DMSO in a 25 µl reaction mix).
Using a Hotstart-Enzyme (or doing a Hot start manually) might also help.
However, if you still have difficulties you should think of designing new primers to get rid of unspecific bands.
I hope this helps
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