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Hello everyone, I need help with my PCR. I am new to the PCR and my aim is to detect the expression of specific genes by RT-PCR. I have enough total RNA (detected by gel electrophoresis, nanodrop) but after trancription I cant detect any cDNA. I dont know where I go wrong since I add all the reagents (random primers-70 C for 10 mins followed by 5 X buffer, 0.1M DTT, dNTPs and superscript II- 42 C for 50 mins).I would be happy if someone could help or give suggestions. I did RT-PCR and detected no amplification whatsoever. I also ran a gel and detected no visible bands. So I assume no cDNA
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It may be that the salt concentration in your initial RNA sample is too high and inhibitory to the RT enzyme. I would purify the RNA with a column and then proceed to RT. -
Assuming you did a good quality RNA isolation. Did you do a 25degree incubation before the 42degree for 50 minutes? Otherwise the random hexamer will not bind efficiently and you will have limited synthesis.Comment
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