I've been trying to run PCR on blood infected with Plasmodium which has low levels of the parasite. The PCR target is a gene of Plasmodium.
Has anyone had experience with this low parasitaemia causing non-amplification? I have tried different DNA extraction kits as well as a range of thermalcycling conditions, gradient PCRs, etc.
I have tested the primers in silico and they work, they are also from published literature.
Any advice would be appreciated
Of the dna extracted from the host only a tiny amount will be from the plasmodium. Hence theoretical pcr works well but the product is still invisible due to the low amount. Try designing either fully nested or hemi nested primers to do a re amplification ( 1/100 dilution of first amplimer) using 20 cycles for the second amplification. The quality of the nested primers is not very important since the template is hugely enriched for the target sequence.
aggree with Dr Paul.
and if you concern about the inhibit factor from the blood, you could try neb Hemo KlenTaq. it works even with 10% blood.
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