i have done RNA extraction and cDNA for tobamovirus then i made a PCR reaction using primer pair Tob uni1/Tob uni2 according to the protocol in Letschert et al., 2002 ( annealing temp. 60C for 45s )but it gave me several bands not in the predicted size .
Can i ask What might be the problem ?
note: picture of running gel of PCR is attached (lane 5 is the result of my PCR)
Regards,
Hello Yasmeen,
Assuming that your primer designing is correct.
There may be several reasons for non-specific amplicon bands. Some of the most common reasons include, incorrect annealing temperature (you can try to increase the annealing temperature); incorrect Mg+2 concentration and incorrect template concentration.
But there can be other causes too, like, premature replication (for this, you can prepare your PCR reaction mixture in ice and then do PCR in a thermocycler which is preheated). Sometimes excess primer concentration can also pose some problems.
But sometimes, incorrect product size may also occur due to nuclease contamination.
Hope I am able to help you partially,
With best wishes,
Madhab
Your main problem looks like degradation of RNA
Have you run your ~ 1ug of RNA on a 1% agarose gel (with 10% formamide + 2 min denaturation @70C followed by ice for 5 min) to check that what you mostly see are the rRNA ribosomal subunits (18s + 28S) and not basically a smear ?
Dr.Hall,
I run the RNA on agarose gel without formald. And it appear as thick and broad band with some smear but the band was obvious and the purity was high.
Then i made the cDNA and it gave a sharp band (pic. of cDNA gel is attached. The first 2 lanes cDNA were made using oligo dT and specific primers . The second 2 lanes cDNA using specific primers only )
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