RT-PCR product- no band - (Apr/11/2013 )
I'm working on Grapevine Leafroll Associated Viruses. I'm using a multiplex PCR protocol, but it doesn't work, even the positive controls. What could be the problem? If you have ant idea about this, please inform me. Thanks in advance.
You have not provided any details. If you want useful input, provide a very thorough description of what you did (reagent concentrations, cycling conditions, primer information).
cDNA synthesis: 1μg of total RNA, 200 units of recombinant MMLV reverse transcriptase, 100 units of RNase inhibitor, 0.5 mM dNTPs and 2.5 μM random nanomers, this mix incubated for 50 min at 37°C. sRT-PCR mix(25μl): 2 μl of cDNA, 2.5μl PCR buffer, 0.2 mM dNTPs, 0.25 μM each primer, 1.5 mM MgCl2 and 0.5 units of Taq polimerase. Cycling conditions 94°C for 4 min followed by 35 cycles at 94°C for 30s, 56°C for 45s and 72°C for 1 min. mRT-PCR(25μl): 2 μl of cDNA, 3.75μl PCR buffer, 3mM MgCl2, 0.3 mM dNTPs and 1.5 units of Taq polimerase, 0.08μM 18S rRNA, 0.8μM GLRaV-2, 0.6μM GLRaV-3, 0.3μM GLRaV-1. Cycling conditions 94°C for 4 min followed by 35 cycles at 94°C for 30s, 50°C for 1 min and 72°C for 1 min and 30s.
Unless I am missing something, I don't see a problem with your setup.
Make sure your reagents are good. I assume you have used these reagents (minus the primers) successfully with something else. If not, test your reagents with template and primer that have had positive results in your hands or in your lab.
Beyond that, especially if you designed the primers yourself, make sure each primer set works by itself before combining multiple primer sets in the reaction.
Thank you very much for your advices. I have tried everyting so far, but none of them worked. I keep triying until I have a positive result, because that's theonly thing I can.