Problem with PCR amplification- high 3' stability - (Feb/08/2012 )
I am trying to amplify a product by reverse transcription PCR. The primers that we designed have restriction sites at the 5' end. The left primer is 21 bp and right primer is 20 bp. On analysis the Primer 3 output says Right primer is unacceptable: High 3' stability. We are expecting a product of 1517 bp but are getting products below 1000bp, 3-4 products depending on the annealing temp. Suggestions pl..
High 3' stability means there are many C/G pairs at the 3' end of your primer, that may cause nonspecific binding of only the 3' portion. Try to redesign the primers, so there are more A/Ts at the end.
But 20 bp for a primer with restriction site seems quite short (maybe someone corrects me), that's the length of normal primer. If you added like six basepairs for restriction, it means your primer is only 14 bp long. Shorter primer means more nonspecifities. You should design normal primer, maybe in primer3 and then add the restriction site sequence on it, it would be longer, but actually only the complementary part does the binding on template, so it's OK.
Thanks - the 21 bp and 20 bp are without the restriction sites. Actually we did get amplification once - but we could not clone the product and now we are unable to reproduce it - the quality of RNA/cDNA is good as we could amplify other products using the same template.
You can try to optimize the reaction. Seems that you are getting nonspecific products, so increase anealing temperature, change magnesium concentration or try DMSO or other additives.