RE-PCR - (Apr/08/2012 )
hi, im trying to clone a gene from the malaria parasite using infect blood spots dried on filter paper as source of parasite DNA. the extracted DNA does not show on gel (due to very poor yield i think) but on using primers specific to my GOI , im able to get an amplification on doing the 1st PCR. however this band is not bright enough and may not lead to efficient ligation with the cloning vector. so i gel elute the 1st PCR product and go for a 2nd PCR using this 1st product(jus 1ul was used) however i get a slight smear in the 2nd PCR thought my product is substantially amplified. so my question is whether my strategy of using a 2nd PCR to get good amplification is correct? are there any chances of non specific products ligating to my vector? should i go for nested PCR instead?
In my experience, nested (or semi-nested) pcr works much better than a second pcr with the same primers. For malarial genes, having very low GC content, you may find lowering your extension temperature (and extending extension time) to help in amplification.