I am using FFPE extracted DNA for analyzing patients with EGFR mutation analysis. In EGFR, I am amplifying 3 exons, exon 18,19 and 21. I am able to amplify exon 19 and 21 but am not able to amplify exon 18. I am facing this problem since last 8 samples I am handling
before that it was working fine. I am using nested PCR approach for amplifying both exon 18 and exon 19. In Exon 18 PCR , I am getting
smearing and shorter non specific bands instead of a clear band.The
PCR protocol that I am using is as follows- 1X Std Taq buffer, 0.2uM
each primer, 200uM dNTP mix and 2U Taq DNA polymerase and cycling
conditions are 5 min at 95°C, 35 cycles of (95°C for 30sec, 57°C for
30sec, and 72°C for 45sec) and final extension at 72°C for 5min. PCR
product size is 400bp with outer set of primers and 380bp with inner
set of primers.
The older FFPE extracted DNA samples are still working with these set
of reagents and under the same PCR conditions mentioned above.
I have tried with all new set of reagents, new primer sets. I have
tried this PCR multiple times by changing Tm,adding DMSO, diluting the
DNA sample, changing primer concentration, dNTP concentration, and
also by changing the MgCl2 concentration in the increments of 0.5
(2mM, 2.5mM, 3mM, 3.5mM, 4mM). I would like to know what could be
causing problem in amplification.
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