I used two samples one is infected to HBV and another not. when I had checked the Real-time amplification data it showed HBV specific primers amplification in both samples infected or non-infected while its amplification should be only from the infected Sample.
Reaction volume = 6uL
( Sybr Green =2.5 ul, primers = 1uL(10uM), template = 1uM and ddH2O = 1.5uL)
Reaction condition ;
Initially 95 degree for 10 minutes
Denaturation at 95 degrees for 15 sec
Annealing at 56 degrees for 1 minute
Extention at 72 degrees for 30 sec
Melting Curve ; 95, 60, 95 degree for 15, 60, 15 sec respectively.
Primers Tm between 59 to 62 degree and Product length from 100 to 150
hi Jitendra Kumar
your protocol seems to be fine but are your primers specific to EBV? clean and separated DNA/RNA extraction? an other thing, since virus as EBV can be very volatil, are you sure that EBV did not infect the supposed controls?
fred
Dear Frederic,
Thank You for your kind reply.
I done primers blast with NCBI according to the result of primers blast primers were specific for HBV.
I confirmed the infection by HBsAg in the supernatant of the experimental plate is positive for that Ag, but I didn't check control cells, so I am not sure for that one.
can you explain other things in detail which should be considered
I will look forward to hearing from you.
Hi Jitendra
if you designed and checked primers with primer-blast, which is fine, the only remaining thing is samples and contaminations. you must therefore check or simply change all control cells, and operate growth and extraction separately (not the same day, not with same pipets or so on, not in the same culture chamber or oven, in different rooms...).
have you ever done PCR with blanck (just water as template)?
all the best
fred
Dear Frederic,
Thank you for your early reply.
Yes, I was every time took negative controls like Primers with water, template with water, SYBR Green with water, SYBR Green with Primers and SYBR Green with template alone, in these all wells result was undetermined like expected or fine but the problem is only the amplification detection in Control.
Thanking You
Regards
Jitendra
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