I am standardizing pcr and melt conditions for a particular exon. The fluorescence intensity of my amplificon is low, when compared to my other exons (picture below). Can I consider such melt curves? There is another peak at around 55 degree C which I think is the primer peak. I have done a temperature gradient, primer gradient, Mgcl2 gradient and also increased the extension time. With all these variations the pattern of the melt peak is as shown below. Is further standardization required?
it is not advisable to consider such amplifcation. perhaps u need to do more optimization.
Definitely not a useful assay in its present form. My preference would be to design 2-3 additional primers in each direction and test all combinations.
You can also try betaine, it worked magic on a few difficult amplicons we had in the lab.
Is there anything particular about the amplicon's sequence? GC content? Repetitive sequences?
Thank you Anna Git for your suggestion . Will try betaine. This amplicon has a high GC content. I am not sure if that explains the melt curve
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