wierd rt-pcr - (Aug/29/2008 )
hi,
ive been using the one step rt.pcr kit for amplifying viral rna, and it has been working for one set of primers, the "easy" ones. the person who was in charge of me before said he used to it using two steps, first making cdna and then using that for a pcr, because the virus we are amplifying is "picky".
nevertheless, i tried this and instead of getting the 0,8 kb fragment i got before with the "easy" primers, i got a giant band, plasmid sized.... tried again, same thing. i used gene specific primers to make the cdna, i will try again on monday with random primers, but anyhow, i find this very fishy adn i dont know what to think.
any suggestions?
thx.
I would prefer to use two steps for RT-PCR. In RT step, I generally use random primer. And then use that as template for PCR. No any problem. Good luck.
ive been using the one step rt.pcr kit for amplifying viral rna, and it has been working for one set of primers, the "easy" ones. the person who was in charge of me before said he used to it using two steps, first making cdna and then using that for a pcr, because the virus we are amplifying is "picky".
nevertheless, i tried this and instead of getting the 0,8 kb fragment i got before with the "easy" primers, i got a giant band, plasmid sized.... tried again, same thing. i used gene specific primers to make the cdna, i will try again on monday with random primers, but anyhow, i find this very fishy adn i dont know what to think.
any suggestions?
thx.