Posted 20 June 2009 - 06:08 AM
I've a question about an oddity that appens when I perform PCR experiments with some cDNA.
I've synthesized 4 cDNAs from the RNAs from 5 different cell lines using random hexamers, now I'm performing some PCR experiments to verify the presence of some RNAs I'm studing. The number of loci I'm studing is 6, for each one I've 2 primer pair, one of which is a nested primer pair. 2 of the loci are even positive, each time I perform a PCR I obtain a fragment of the expected size, but the results for the other four are not always the same!
When I performed the PCR for the first time I obtained a results, the second time I did the experiment using the same condition the result was different! Some of the samples that amplified the first time didn't amplify during the second experiment, other samples that didn't amplify in the first experiment amplified in the second.
I tried performing the experiments using the nested primers too, and sometimes the results obtained were different from the ones obtained using the external primer pair.
Did someone experiences this oddity?
Is is possible the RNA I'm looking for is a low copy one so the number of the First Strand cDNA molecules is so much low that sometimes the PCR reaction fails?
Posted 21 June 2009 - 12:45 PM
Posted 21 June 2009 - 01:16 PM
Posted 21 June 2009 - 08:17 PM