Possibly you have some primer dimer contamination of a reagent or pipette or working area from a previous amplification. Using a new aliquot of primers from unused stock and borrowing another researchers pipettes and pcr reagents set up the pcr again including a no template control sample ( water) . Ideally using a hot start enzyme. If the problem disappears then you will have to do a clean up of all equipment and replacement of all reagents and your working area

Hi, Kashaf Javed and Paul Rutland

As always, Paul has already given you a good possibility. So, I will approach by other way.

1) Have you chcked your new samples for amount (Yield) and quality? Have you done anything different? For example, DNA or RNA extraction with a different extraction/purification kit? Anyway, check its amount and quality.

2) Does any of your kits are older or may have suffered temperature variations?

I know you said everything was just the same but, usually, even more If it was a long time ago, our memories did not recall every single detail ... So, check it once more.

Hope it may help you!

Paul Rutland

thank you i will try this again, well i have used a new aliquot of master mix, and always make new working stock of primers for my every pcr.

It is good to make a new working primer stock Kashaf Javed but are you making it from frozen aliquots of stock primer or always from the master stock as frequently freeze/thawing primers can cause them to degrade and stop working.Primers will also stop working if they are dissolved in water not TE as water becomes acidic and depurination can take place

Paul Rutland

thank you for your recommendation

Hi,

Ideally, do a bigger solution of primer silution for use and divide it in small you're going to use for only a few times. It's much better and always in TE instead of water as Paul Rutland said.

All the best.