I have gel eluted my 3'race product.Upon subsequent dilution of the gel eluted product and reamplification using nested primers (nested gene specific primer and nested universal primer supplied by the company), I have been getting numerous bands for 3'race pcr alongwith my band of interest.The sample run on a agarose gel looks like a ladder of DNA. I also tried using nested primers internal to the sequence and i get a clean single band apart from the primer dimer.
I have tried playing with temperatures, tried Touch down pcr and got fresh stocks of primers to sove contamination issues if any. The problem seems to persist.
Can i have any suggestions please?
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3' Race PCR
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