I use the primers synthesized by molbiogen and all the pcr components purchased freshly and cDNA synthesized shows expression in housekeeping gene. I tried in normal DNA i got faint bands for the other gene. So please can anyone suggest the possible reasons.
Well Generally these can be rectified by adjusting the following:
* Reduce polymerase concentration. * Shorten extension time. * Reduce total number of cycles. * Increase annealing temperature or try 2-step protocol. * Optimize Mg2+ concentration. * Lower primer concentration.
Reduce your template – Having too much template seems to be the most common issue. Try to reduce your template to see if that improves your results.
Lower cycle times – This is especially helpful when your template concentration is higher. Keep within 20-35 cycles.
Reduce extension times / Raise annealing temperature – both of these will help improve your PCR results by reducing the occurrence of nonspecific binding and smearing bands.(Adding DMSO upto 5%helps)
If not, your template may be sheared or contain exonuclease. Use a fresh template.
Hopefully this helps,
Cheers.
Hi Amit,
maybe I'm wrong but when you said that you tested cDNA and followed DNA analysis, maybe you missed something.
cDNA are representing only expressed genes and their sequences are composed of only exons and UTR. when designing primers for RT-qPCR, one of the primers need to overlap a splicing site to avoid DNA amplification (since you'll every time have DNA in your RNA and cDNA preparations). then you can understand that using primers dome for RNA will interfere when testing DNA.
fred
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