I have two viruses that are fairly similar. The design of the primer relied basically in minimum differences since these viruses are too similar. I could make the assay work under SYGR assay but after 30 cycles some amplification is detected. When I run the gel, no bands are visible. We decided to move to Taqman probes to eliminate the detection that SYGR has. However since the viruses are too similar, the probes are not specific. However the set of primers are like 300bp apart. Do you think I might have a problem if I try to multiplex the sample?
Hi
The ampilcon of 300 bp is too long for aTaqMan system. check your SYBR primers with a suitable software like nucleotide analyzer for primer dimers. you may check the specificity of your primers using conventional PCR, if you obtain two different band then you could decide about applying melting analysing methods.
Hi, although the idea length of amplicon is 50-150bp, but I think 300 bp is also could work. Based on the specificity, I want to say whether you could design the primer beyond the same sequence. Do you have the genomic sequence of these 2 viruses? Before you run RT-PCR, you may run normal PCR first to check the specificity. I strongly advise you to change the primers and run RT-PCR with SYBR. Good Luck.
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