Melt curve analysis - (Mar/10/2006 )

Hi!

Has somebody got an explanation for this melt curve analysis of plasmid dilution series and unknown DNA conc. The certain small peaks were obtained by the lowest dilution of plasmids.

Primer dimers or unspecific products? The Primer conc. was 15pmol, which was done for the first time in this run (25pmol before) and I changed form 3-step to a 2-step cycle.

Thanks,Sil

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Put the product on a gel and you will see...

(I guess its nothing serious)

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I didn' t see any specific bands on the gel. And this peaks occurred actually for the highest dilution, not for the lowest. 10^0 (lowest=10^6)

So, does any body else has got a good idea?

Thanks for your help, Sil

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only in the highest dilution? then it makes the most sense that you're seeing some mispriming and a little bit of nonspecific product

again, though, I agree with Ratiopharm..the relative amount is very tiny...if you don't see specific product you are probably OK. are your results reproducible?

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My results were optimized, because my efficiency, which has been always too high, was about 100% in this run. I reduced primer concentration (25pmol-15pmol) and did 2-step pcr with 60sek annealing instead of 3 -step before,where I got too high efficiency but nice melt curves.

Should I reduce the annealing instead and try 3-step cycling with 20sek?

Thanks for your comments anyway, Sil

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