When I visualised the band on the gel electrophoresis, there were not many different changes when compared to CDNA product using normal PCR technique in terms of the size of the band. But I noticed that the intensity of the CDNA band is more dense than ssDNA (Asymmetric PCR). Can we confirm that the less intense bands are the results of ssDNA generation from asymmetric PCR? Is there another methods which we can use to confirm the ssDNA -pcr product other than gel electrophoresis?
The ratio of primer Forward:primer reverse was 1:10 for asymmetric. Symmetric (1:1). other parameter is fixed.
As in an asymmetric PCR the amplicons increase in a linear fashion and not in a logarithmic fashion the total amount of product formed is much less than in a conventional reaction so the product will appear as a faint band on the gel compared to ds DNA product. But you can actually quantify the amount of product you produced and see if it matches your calculations. For eg. if you started with 5 ng template at the end of 10 cycles you should have ~50 ng ssDNA. A Nanodrop or Bioanalyzer could b used to quantify the ssDNA.
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