Hello,
I have designed a pair of primers to amplify a part of an endogenous retrovirus that is present in all individuals of the target species with each individual having several copies.
Im using a degenerate position in the Rv primer to amplify all the variants, but what is happening to me is that if I do the PCR in identical conditions with the same sample in 2 different days I sometimes get a product band while others I dont.
This is extensive to all the genomic DNAs I use and I checked the integrity of those DNAs by gel and amplifying COI.
Also, in addition to sometimes amplifying and sometimes don't, I sometimes get unspecific bands and later I don't.
I checked and they dont have hairpins or form dimers.
I noticed increasing the number of cycles from 30 to 40 increases slightly the number of samples that amplify each time.
The reverse primer has a low annealing temperature of 49ºC indicated by the manufacturer but I found that it works better at 51ºC (higher product yield).
Any idea what might be happening?
- Have you please checked gel loading error means gel well has not filled properly by PCR product?
- Changing Annealing temperature may resulted in getting unspecific bands.
- In case of reverse primer you have got better result at 51ºC though manufacturer indicated 49ºC. Sometimes we need to standardized manufacturer products in our lab.
In addition to the good answers above it seems to me that you have one aspect that is just on the verge of working and even a slight difference in efficiency makes a huge difference in final product. It may be that your dna has some pcr inhibitor present and the slight pipetting differences make a difference to working/not working so you could try increasing the MG concentration and using slightly less DNA sample
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