Hi, everyone I'm working on the detection of mutation done by CRISPR.For this, I'm using the Surveyor Nuclease Assay.I'm able to see a clear band after the PCR.However, after heteroduplex formation and treatment with Surveyor Nuclease, I'm getting smear bands.I'm using the PCR products directly for heteroduplex formation? is this correct. The kit doesn't have any duplex buffer.Any advice for duplex formation followed by treatment with Surveyor Nuclease. (Does it need 2microliters of Nuclease instead of 1)
Thank you and appreciate your time.
There is a good protocol for heteroduplex formation on page 14 of the attached guide. Concerning smears surveyor has 5'exonuclease activity as well as ssDNA nicking so smears are common and minimised by the addition of extra Mg ions as in appendix A in the attached.
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