can't get rid of DNA contamination even with DNase for RT-PCR - (May/10/2005 )
i extracted my RNA using TRIzol and there was DNA contamination. so i treated the RNA with DNase (RNAse free, from Roche) by adding 2ul of DNase into RNA sample and incubate at 37oC for 1h. DNase was then inactivated by incubating at 70oC for 15min. Afterwards, i ran the gel for the purified RNA and i did not see any bands for genomic DNA, so i did my RT-PCR. unfortunately, i also got strong bands for negative controls, which were RNA samples without reverse transcriptase. Can anyone help me on this. It is quite anonying!!
How old are your DNase reagents? I use the Fermentas product (cat # EN0521) and have always had good luck.
Also, I design at least one of my primers spanning two exons so that the genomic DNA will not amplify (unless the intron is very small). Good luck.
My DNase is quite new. I bought it las year. I think maybe my PCR reaction is contaminated, so i am doing several controls for my PCR to see if i can get anything without DNA template. Thank you for your advice!!
How many freeze-thaws has your DNase been through. More than three freeze-thaw cycles can affect DNase activity.