I am working on a PCR. I did gradient PCR to make sure that the annealing temperature mentioned in the reference study is the best temperature for annealing. I found there were lots of secondary bands in low temperatures, and these bands disappeared in higher temperatures.Is there any reason for that?
Thanks
When the annealing temperature is lower the oligonucleotides can bind with less specifity to the template DNA. Due to the lower stringency conditions you observe PCR bands that disappear when you increase Ta. Always try to avoid the synthesis of unspecific products (PCR artefacts).
Regards, Christian
it is clear to see secondary bands, and the reason is that your primers anneal to some other sequences which are not 100% match to your primer. for example if the annealing temp for your primers is 59C and you see one sharp band at that temp, this means your primers totally matched to the DNA sequences you have designed for, but when you make the temp lower the primers will attach to other sequences which are less match to them. e.g a 22n primer will totally attach to the specific sequence which you want at 59C, but at 57 C , 18 n of them will match to another sequence too,and this sequence will polymerase too. in this situation the sharpness of bands will be lower.
Hi,
It seems that your primer pairs need high annealing temperature for avoiding non specific bindings.
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