Currently I designed primer by Primer BLAST but even it showed the no non specific band the real time results showed non specific binding
I am not aware of fact whether BLAST take consideration of the non whatson base pairing also in consideration or not.
Is this lack of training based on unconventional base pairing may be the reason why still failures in primer design occur.
Hi Sathyajeet
What was your PCR template? Did you select the corresponding organism in Primer Blast?
Most PCR is performed using ds DNA as template and ds DNA maintains its helical structure by Watson-Crick Base pairing. Most primer design softwares are designed for such PCR reactions and therefore don't include Non-Watson-Crick base pairing.
If your PCR template is dsDNA, please review further details of the primers you designed - Tm, GC%, self complementarity, 3'end bases of the primer, etc. and also review the reaction conditions - temperature profile, primer and template concentration, buffers, number of cycles etc.
Also check if the primers you received from the company are indeed having the same sequence that you designed. Mistakes sometimes happen.
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