MULTIPLEX REAL-TIME PCR WITH SYBR-GREEN - (Oct/15/2007 )
I am working on multiplex Real-Time PCR with SYBR-green for amplification of 2 target genes and one Internal Control. The Tm values that I get in the multiplexed reaction for each gene is different from what I get while running the samples with individual primer sets. I have tried all possible combinations and it still is not working. Is it possible that the presence of more than primer set is affecting the fluorescence or giving spurious readings?
MG
-mshg-
QUOTE (mshg @ Oct 15 2007, 02:09 PM)
I am working on multiplex Real-Time PCR with SYBR-green for amplification of 2 target genes and one Internal Control. The Tm values that I get in the multiplexed reaction for each gene is different from what I get while running the samples with individual primer sets. I have tried all possible combinations and it still is not working. Is it possible that the presence of more than primer set is affecting the fluorescence or giving spurious readings?
MG
MG
Dear MG,
I would say that amplifying 3 target genes at one go using DNA intercalating dye is a bot too ambitious.... :-)
By the way, you may try using Evagreen instead of SYBR green.
-Hadrian-