Dear Inayat
I think Primer binding site or concentration of DNA might be in small proportion reasons for PCR failure. One more thing you should preserve it in double distill water.
HI, which extraction method did you use? Did your positive control in your PCR showed the expected band? Cheers, Nadine
Dear Nandene.Yes positive control done and there was amplification in the positive control...Two methods Quigen Kit and manual protocol(GuHCl & SiO2) method followed.Cheers
On Checking on nanodrop the conc comes between 50 and 150 in old samples.And in fresh it comes up to 400 ng/microlitre.
Have you checked your DNA on a gel and saw it ? And what is your primers Tm and the PCR set up (annealing time, extension time and cycles)?
I am a little bit confused here--
You used 2.5 ul DNA in the PCR, and the DNA concentration is Old = 50-150 ng/ul, Fresh = 400 ng/ul. Therefore, the DNA template in your PCR reaction should have been either at least 125 ng ( 50 x 2.5) from the Old sample or 1000 ng (2.5 x 400) from the Fresh sample. How did you get that 25 ng DNA amount in your answer? Did you dilute the DNA samples?
Dear P.K Panday
I am using the primer to amplify the DNA of mammal and the primers used are that of a reptilia. amplyfying cyto b gene.
I dont collect the feces from the zoo.they were collected in wild.
In my past work i got amplification from the samples but not now...
You can do a positive control experiment by other easy amplication gene segment, such as 16S rDNA, so it can judge whether you DNA is wrong or PCR is wrong.
Dear Cheng
A positive control sample was also loaded,and their was amplification
PCR is wrong, your primer should redesign, use a more conservative region.
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