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non specific PCR
Posted by: jayagosula (IP Hidden, New member, 1)
Date: November 8, 2005 11:56PM
I had standardized PCR for 1.8 Kb.iam getting some non specific bands between 500bp to 1.0 Kb. but later in same pcr conditions , using gel purified amplicon as template, i am getting smear and it is not giving expected amplicon . I tried temperature gradient also?
after gel purifying, i cloned the amplicon . in PCR screening of the clone also iam getting bands around 500bp to 1.0 Kb. . out of 50 colonies i got only 2 positives showing expected amplicon. i couldn't able to understand why it is happening? after gel purification, when i checked small aliquote it is single band moving at 1.8 kb. what ahpuld i do?
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