Same cDNA is used for other amplifications and i m getting result for that ,, actually the Tm of the primers is too low unfortunately it is around 35 degree , when i put an PCR at Tm 28 i got some amplification but of non specific size, but the amplification from gDNA remains same of specific size,, i.e at 28 and at 35 degree.. any solution for this problem???
Do you mean that you can get the gene from gDNA but you get non-specific size using cDNA?
Annealing temp at 28 is too low, leading to unspecific binding. If the Tm is around 35, you can try gradient pcr between 32-40 degree c, it works for me. If it doesn't work, you might need to consider designing another set of primer
did you check that the product from gdna is the correct product by sequencing? The primers anneal at such a low temperature that I am surprised if there was a clean amplification and as Vikki says there should be much non specific amplification. I am thinking that neither the genomic nor the cDNA are working rather than gDNA is working. Primers are cheap. Can you design longer primers?
Dear Ravi Shankar Gautam
It happened to our study later on we figured out that primer binding sequences were between exon and intron junction. You may change primer binding site.
@Paul Rutland I haven't checked that gDNA amplicon by sequencing yet as I m more concern about amplification from cDNA but as u told me that its due to too low Tm I will definitely take this in consideration and will design a new Primer.
Thanks a lot for your valuable suggestion.
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