1. You must check actual size of amplicon by performing primer BLAST- https://www.ncbi.nlm.nih.gov/tools/primer-blast/ (Just put f and r primer in this window, select database nr/nt and wait)/

2. Also check if the primers reported for real time PCR were based on genomic DNA or on cDNA/RNA sequence. If the primers were specifically used for cDNA/RNA amplification, you may be getting introns amplified in genomic DNA resulting in increased size.

1. You must check actual size of amplicon by performing primer BLAST- https://www.ncbi.nlm.nih.gov/tools/primer-blast/ (Just put f and r primer in this window, select database nr/nt and wait)/

2. Also check if the primers reported for real time PCR were based on genomic DNA or on cDNA/RNA sequence. If the primers were specifically used for cDNA/RNA amplification, you may be getting introns amplified in genomic DNA resulting in increased size.

Besides what Yashwant Chavan proposes your primer may also amplify a completely different product in genomic DNA than the one they were designed. If the primers were designed for cDNA which I assume they were, and one of them was designed on an intron/exon junction, what you see on the gel will come from a completely different part of the genome. To answer your final question, you should try the primers on cDNA and if they work as expected use them (if you want to do an expression assay), but be sure that you have completely removed gDNA from your samples. Of course you could always design a new pair of primers that doesn't amplify gDNA.

• In short no you cannot use these primers for qPCR at least with the conditions yielding the unexpected 400bp product 
• As already mentioned verify specificity by BLAST 
• In addition make sure your annealing temp only results in specific amplification by approximating to the melting temp of the primers : in particular annealing temp that are specific but also result in efficient amplification of your specific product tend to be Tm-2C down to Tm-5C
• One possible explanation for your unexpected band is non specific amplification due to low stringency 
• Thus apart from BLAST check try increasing the annealing temp in your PCR reaction by 1-2C 

non specific binding check primer

Thank you Yashwant sir, Georgios Theodorou sir, Laurence Stuart Dawkins-Hall sir and praveen sir

primers were designed from RNA sequences. Out of these genes, one full length gene size is 1992bp and the amplicon size is 92 bp for real time PCR. But it shwoing 400bp band in standard pcr. Thank you for you suggestions I will go forward as u told. Thanks again

Hi Manoj,

I am assuming that you are using primers/probe (Taqman) and not SYBR green. First thing is to go ahead and do BLAST as recommended by others. Are there multiple bands?? And what about the negative control?? If there are multiple bands and band in negative control then it might be due to contamination. I would also suggest you for trying Gradient PCR in order to find the exact annealing temperature. Apart from this please try doing directly Real time PCR with following conditions:-

18 µl of forward primer, 18 µl of reverse primer, 5 µl of probe and 59 µl of water. A final volume of 20 µl, comprised of 1 µl of assay solution, 10 µl of Gene expression mastermix , 6 µl of water and 3 µl of DNA, was prepared for each qPCR sample. The following protocol was implemented for qPCR reactions:  an initial holding stage of 2 min at 50 ͦ C, followed by the cycling stage of 40 repetitions of each of (1) 15 min at 95 ͦ C, (2) 1 min at 60 ͦ C.

I have used this protocol many times and it worked well for my Real-time PCR experiments. 

Actually gDNA has no role to play. You may be adding more primers in the PCR experiments and that is causing dimer formation.

• I'm going to concur with Yashwant as well:
• Primer BLAST is an excellent tool for making primers that specifically target exon exon boundaries
• If all else fails make new primers but design primers that cross exon exon boundaries using primer BLAST. They are your best primers for real time qPCR
• I attach an SOP I have devised for making and optimising real time qPCR primers
• I hope this at least helps illuminate qPCR primer design considerations