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RT-PCR - cDNA synthesis (Aug/26/2004 )

Hi, i just started some RT-PCR experiments on a Sphingomonas strain (High GC content). I can get a good RNA preparation. After DNAse treatment i still see the 16S and 23S bands. Unfortunately i can get no cDNA synthesis. I use random nonamers as primers and 48° as hybridization temperature. Maybe someone can give me a little help.



How do you know you got no cDNA synthesis, by PCR or electrophoresis? If by PCR, make sure your PCR part works well; if by electrophoresis, you should see a smear of synthsized cDNA of different size. If not, make sure that every component of RT reaction is OK. How long your RT goes at 48C? We usually use 42C for extension. You may want to try 42C. Another suggestion is do not use DEPC-treated water in RT reaction as DEPC may inhibit the RT and PCR reactions.