I made cDNA from plant RNA viruses. If I run PCR within the day, everything worked perfectly. I got positive control as positive and of course negative control as negative. But if I store cDNA at -20oC and run PCR next day or so I do not get amplification of positive samples. I would appreciate any feedback. Thank you!
Could be nuclease in the cDNA that takes time to cleave up your cDNA. Could even be that the cDNA 'breaks' with freeze-thaw due to pH change --depending on what it's in (water? buffer?) and whether your sample is in a 'frost free' freezer? If so, and your sample tube is simply in a cardboard box, then fluctuates in the surface temperature of the freezer compartment result in 'freeze-thaw' cycles. So put you sample tube in one of those 'bench-top' coolers, which would insulate against such freeze-thaw. Also consider secondary structure of the cDNA that fails to dissociate after freezing or even a nuclease formed by the cDNA or more likely RNA (RNAzyme).
Hello Nancy thank you for your answer. All the reagents related to RT are from ThermoFisher (SSIII) including water and buffer.
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