I am trying to amplify a region of 287bp from human genomic DNA. Initially I got the desired band but after the lockdown period I am not getting the band. What I am getting is either blank in gel image or smear. I have tried everything from using gradient of each component at one time to replacement with new components. But I am not getting the result. Kindly help me regarding this. What could be the reason?
PCR buffer = 2.5ul, dNTP mix = 0.5ul, MgCl2 = 0.5-3.5ul, FP/RP = 0.2-0.5mM (final concentration), Taq Pol = 0.2-0.5ul (5U/ul) DNA template isolated from cell lines = 20-100ng
Fig showing bands is the one which I got initially.
I would say you are putting to much DNA template. But if you say you are putting 20-100ng should be ok. Just to discard, I'd try some dilutions.
Usually if a primer pair stops working after a period of disuse, chances are that it has degraded. Was it stored in TE buffer ? If so, you can set up a touchdown PCR starting with a temperature just below extension temperature in the first cycle and one degree less in each subsequent cycle. If the primer pair is intact you should be able to see the original band, albeit with lesser intensity than before (because TD-PCRs do not allow for high yield when compared to simple PCR cycles). If you don't get a band by TD-PCR, it is best to reorder the primer pair and start again.
If you do get a band, then use a tiny amount of the product of the TD-PCR as a template for your usual/regular PCR.
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