I am doing fourplex mqPCR panel for pathogens by Taqman probe . I did well choose for different dyes option (reporting dyes). real time PCR is five channel 7500 applied biosystem.
For primary checking, I added all of primer and probe with accurate concertation and just put one template (recombinant vector) but in result amplified three out of four primer/probe. It mean, amplified not only in target template but also another pathogens that I put primers and probe.
Is that cross interference?
What is the idea to solve this condition?
When you run PCR with a no template control ( water instead of dna) do you get any amplification?
I put four primers/probe reactions and one template (plasmid vectors) in each well and tested for four. 3 of them amplified well only their related template and base pairs were also clearly noted.
Just one well noted 3 amplification even though that I used one template.
but when you amplify with water innstead of dna do you get any amplification? If you do then you have a reagent contamination
There are reagent contamination in your PCR system. In addition, you'd better perform specificity evaluation of your primer - template (recombinant vector). Primer pairs having target regions in the template may lead to nonspecific amplification.
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