RT-PCR problem - negative control (Aug/24/2005 )
I was wondering if any of you could give me some ideas how to solve my problem concerning RT-PCR. So Im working with human RNA from virus and Im using Promega Kit for my RT reaction but since I started using new primers(300kb) in my PCR I get a band for negative control were nothing should appear since Im not adding RT enzyme to the sample. I repeated that many times concerning my mistakes or contaminations in the stuff Im using. Then I thought that maybe my RNA for positive and negatie control is cotaminated.Therefore I treated it with DNase and then carried RT reaction and 2 step PCR afterwards. Unfortunatelly negative band appeared again. What is very strange is that when I used the same RT reaction for PCR with different primers(600kb) neither negative control nor PCR product is observed, the only one is positive control!
Does anyone know what I should do to get rid of this negative control and what can be the explanation for my problem?
I assume you are refering to a 300 bp pcr product for your reactions. What percentage gel are you running to analyze your samples. You should run a no input control to check for contamination or primer dimers. In that size range, you need to run a high percentage agarose gel to separate low mw bands.