I have run PCR of my control and treated samples by using primer of nitric oxide synthase gene at 56, 58 and 60 degree centigrade. After amplification no band appear in control sample at 58 and 60 degree and two bands (one specific size and other non-specific) appear at 56 degree, while product of non-specific size appear in treated sample at all mentioned temperatures. What could be the possible reason?
Hey Kinza Rafi,
there could be a few possible reasons. One interesting thing would be the fragment sizes of your actual amplicon and the one which is non-specific. Furthter, do you use gDNA or cDNA? Do you want to run pcr for expression analysis? Also, Primers could have Introns (maybe make sure, how you design you primes for the gene, at gDNA level, or only CDS?). The sequence of your primers would also be fine to see to say more about problem with annealing temperature. You can also try a gradient pcr to get your specific annealing temperature.
Good luck!
Hi Kinza,
Annemarie has provided a nice suggestion using a gradient PCR, in addition there is need for you to answer some of the questions raised by her. For instance, if the non-specific band is of a very low size then it may be a primer dimer, you might need optimize or redesign the primer to solve this problem. But if the nonspecific band is not a primer dimer then optimization and several gradient PCR may help in solving it, if it still persist you may also reconsider designing another primer. To check the quality of your primers you may check below link
http://www.bioinformatics.org/sms2/pcr_primer_stats.html
I have designed primer for complete cds to analyze the expression. I have also tried gradient PCR but the result is same, at every temperature the product size is greater than the actual one.
What is the product size and what is the actual size. If possible you can give the accession number of the nitric oxide sythase gene you are trying to amplify.
The actual product size is 150 bp and I am getting the product of above 500 bp. The accession no. of gene is GenBank: L02881.1
- Badges
- Science method