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?
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.