Hello,
To check specificity of your primers by ncbi blast, we find identity percentage, query cover and E-value as the parameter to be taken care off. I know that query cover is important but i am getting unintended targets when i blast the primers with less than 70%, My question is can I not consider those unintended targets with less that 75% query coverage?
Also, if the unintended targets have higher e value can I omit that?
If the unintended targets are from different organisms or cell lines than the one you want to target and you are using only one organism or cell line then I think you can ignore them because there would be no unintended targets in your samples.
But if the unintended targets are scattered around your target genome then you will get a lot of non specific bands.
In both cases you will have to test the primers empirically.
I hope this helps.
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