I have been dealing with degenerate primers for paramyxovirus virus detection. The detection assay calls for a heminested PCR with the same reverse primer used for round 1 and round 2 of the PCR reaction. The issue arises when trying to perform round 2 using the round 1 PCR template.
I wanted to ask if there is some specific way to dilute/ purify the round 1 PCR products to get proper bands in round 2.
Also, i did not experience this problem when dealing with Nested PCR primers that are designed differently for the forward and reverse reaction.
You are probably getting a smear of many size dna because of over amplification. Transfer of the outer primers does not help so dilute 1ul of first round product in 100ul of water then re amplify 1ul of this diluted dna using a 24 cycle pcr but remove one pcr tube at 14, 16, 18......up to 24 cycles . Some of these tubes will have clean and strong amplification and will be a guide to second round amplification in the future/ Remember in pcr every 10 cycles is 1000 times more product so over amplification is very easy in nested pcr
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