what are the annealing temperatures of the primers and what annealing temp is the pcr using?
Are you getting a fuzzy band of 50-100 bases in your pcr?
Can you attach a gel picture?
Do you mean that all primers are run in a pcr with the annealing temp at 52c?
What are the calculated annealing or melting temperatures for all of the primers please?
Have you tried any lower annealing temperatures in the pcr and have you run the internal and external primers separately and did they work at 52c?
Yes al the primers are being run at same temperature.
I used gradient PCR to standardise my annealing temperature. i was getting results at 51, 52 and 53c.
It is hard to say what the gel image tells us without a ladder but the smaller band looks like a primer dimer which might be removing primer from the mix and leading to non amplification. If this is a 20ul pcr volume I would try half the amount of dna and with annealing at 51 run all combinations of primers so outf-outr, innerf -outerr, innerR to outerF and innerf-innerR. If any of these produces a strong primer dimer then you may have to use a hot start polymerase and less primer in your assay. If your lab does have a hot start polymerase then try the 4 primers and reduced dna and 35 cycles of pcr anyway
I run arms pcr where control band are clearly visible but allele specific are not visible .I add DMSO also , but no such result are seen
Fresh primers , with fresh master mix is being used still not working.
Can anyone please suggest
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