MSP product looks like some smear

[Amily]

Hi, I am trying to identify the methylation status on my Gene. The PCR product is around 400bp. Based on the result of agarose gel, it looks like there is some smear except the major band. I send the samples to sequencing. The background is super high. After I did gel extraction, the sequencing data is readable although the background is still high. Is it possible that the product is too long? Or maybe the primers are not specific? Any one can give me some suggestion? Thanks

[danfive]

400 bp is not too long for sequencing. Are you cleaning up the PCR product prior to sequencing?

[admin]

400 bp is quite short as danfive mentioned. The problem may be as danfive mentioned that you are trying to directly sequence your pcr product?

Have you tried pcr purification or subcloning into TOPO vector then sequencing?

cheers