I have some problems with nested PCR for low viral load- HBV patient sample.
Kit is Expand High Fidelity PCR kit from Roche.
Samples is total NA from serum of HBV patient sample, extracted by MagNA Pure 96 system. Viral load is from 105 -109
I got not bad results in high Viral load108 -109 However, the lowers are not detected (Picture 1).
In addition, I got dark smear.
The annealing temp. is 67-68 oC
Please take a look at electrophoresis photos and give me some advice for improve the result.
I also try with gradial annealing temp. from 55-68 oC for medium viral load sample, but it did not improve.
2% DMSO add to reaction mix also did not help (Picture 2)
Thank you so much for your advices.