Are these new primers or dissolved in water not TE?

Does this primer pair work on its own to produce the band?

Do you have a primer dimer fuzzy band in the multilex reaction of size less than 100bp

What polymerase are you using?

What is your extension time?

have you tried increasing the Mg concentration

If the primers give you the large product when NOT multiplexing, then the issue is with multiplexing.

Start like Paul Rutland says & try testing that pair of primers as a stand-alone. I assume you have a good positive control that you know will give you the 1791 bp product.

Let us know what you see!

Dear Paul Rutland thank you for your response I resuspended them in PCR water, and I am using the My Taq HS Red mix and did not try the pair alone I will try it also and there in no fzzzy band below 100 bp, infact I see the aggregation of primer at the extent to the which it travel. The extension temp is 72C for 2 minute in pcr cycle and also final elongation. it is a prepared master mix so can I increase the Mg in that case. I am anxiously waiting to hear from you.

Dear Katie A S Burnette thank you for your response I did not try the pair alone but I am going to try it and yes I have standard strain which is used as positive control in assay.

Good that there is no primer dimer. I prefer diluting and dissolving oligos in TE to keep them alkaline and to inactivate nucleases but if the primers are quite new this will not be a problem. You can add extra Mg when using mastermixes but I would try normal mastermix with an extension of 2.5mins and if you get a band then try the multiplex on the sample with 1mM extra Mg which can often help when using multiple primers ( which can remove MGg) and also when the dna has some pcr inhibitors.

good luck

Paul

Tm

Dear Paul Rutland once again thank you for your valuable comments, I am going to try with these tips and will see the results.