Problem in RT-PCR - getting a band in the negative control (Jul/05/2008 )
I'm doing RT-PCR experiments on some immune genes, with actin as the positive control. I extract RNA with the Qiagen RNA easy kits, with a DNAse treatment on-column and another treatment off-column. I use 2ug RNA for making cDNA (of 22ul), using the promega AMV RT kit. And use 2ul cDNA to do a PCR for the genes of interest with the GoTaq green PCR master mix (promega). My primers are 16-20bp with an average Tm of 55C, and they all span intron exon boundaries. My problem is I'm consistently getting a band (of the same size as expected products) in the noRT control.
The bands are certainly not gDNA, as the gDNA amplified product is going to include the introns as well. I've also confirmed it by doing RT without DNAse treatment. I have run a control with just the primers and PCR master mix, and get a band there I then got the suspicion that my primers may be contaminated and so got a new primer, and STILL get the band. I ran another control with just cDNA and pcr master mix, and I get no bands there. My water control is also fine. I've used a new batch RT and PCR kits, and still I get the band.
I'd be really thankful if you could spend a minute and suggest me something.
How do you perform RT: specific primers or something else?
You could sequence the band if you what to know what it really is. Although it will not tell you how it got there.
I use poly dT primers during RT.. And yes! I did sequence the band and found it to be the gene I'm looking for .
please help me.. I'm awaiting your response.. I already lost two months in this
Thanks in advance
Your water control has enzyme, buffer, primers, dNTPs, and is missing only the cDNA? And it is negative, while your no-RT control is positive? Perhaps you have contaminated your no-RT control sample with some cDNA, or with a PCR product. Use barrier tips and an unopened box of tips and plasticware. Avoid autoclaving or handling these as much as possible.