I have to amplify HVR1 of AMA1 in Plasmodium falciparum isolates...Annealing temperature given in articles is 55 o C and i am also following all the conditions but still i didn't get positive results...
@ yinying Yes i dilute the Primary PCR products 10X and then take 2 microlitre of the diluted product for Nested PCR.
Hi,
I would recheck the infos given in the paper a bit. Did you use the same Mastermix? A Gradient PCR would be a good point to start. You could also post the PCR conditions from the publication with setup as basis for recommendations
Thanks
Sven
The cycling parameters for the primary PCR were as follows:
10 minutes initial denaturation at 94°C followed by 35 cycles with 1 minute denaturation at 94°C, 1 minute annealing at 55°C, 2 minute extension at 72°C and a final 10 minute extension at 72°C. The primary PCR product was diluted 10-times and 2 μl of it was used in nested-PCR to amplify the 500 bp region encompassing domain I (or hyper-variable region-HVR) using the AHVR-F (5'-
CTGGAACTCAATATAGACTTC-3') and
AHVR-R (5' TTCTTTCTAGGGCAAACTTTTTC-3') primers.
The cycling parameters for nested primers were the same as for primary
PCR primers except that the extension at 72°C was carried out for 1 minute.
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