The primers which have shown amplification before with some individuals are not amplifying always, while PCR controls showing amplification i.e. not the problem of chemicals; perhaps stems from 'inhibitors, any other advice??
Dear Ray, You have not mentioned what is the time gap? If more than 6 months at -20 deg C or more than 2 months at 4 deg C storage, check for the primer degradation. Just load the stock solution and solution with working concentration side-by-side on ethydium-dye-stained agarose gel (4%). If you find smear or no band, get the primers synthesized freshly. If you see clear band, try using different concentration of MgCl2, different source of water, Taq from different supplier. Once you started getting consistent results.. you use that particular standard for your other samples.. All the best
It is common to have inhibitors especially in your gDNA extract. You can try purifying your gDNA extract further, with a column, if possible. Alternatively, try diluting your gDNA (I usually try a series of dilutions, i.e. 10x, 50x, 100x dilutions) before using it as template for PCR. Good luck!
Dear Dr
I would like to thank you for your question.
Try change the Primer or new isolation of your DNA samples
best reagrds
I would also support diluting your samples to minimise any effects of inhibitors. Try 1/10, 1/100 & 1/1000, often it the really dilute samples that will then amplify well.
Good luck!
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