I am amplifying a target fragment recently. I have done a gradient to determine the annealing temperature, but there are still many specific bands. May I ask you if the amount of template will affect the specificity of the PCR product
Too much template can lead to no amplification if the template dna has pcr inhibitors. It can also lead to amplification of bands from primers finding other binding sites on the genome which might usually be too weak to see but with a lot of template they become more numerous and visible. The type of template is important, You might use 20ng of genomic dna ( about 30,000 copies of the template) but if amplifying a GOI from a plasmid then you have one target every 5000 bases so using 1ng would be plenty of starting template and using too much can lead to over amplification and larger bands than expected as the amplimers anneal to each other and extend to unexpected lengths
The amount of DNA template in a PCR has a negative effect on the outcome of a PCR procedure. Using too much DNA template, results in packed DNA in the confined space of the reaction vessel and can lead to false priming and even poor DNA synthesis due to the obstructed diffusion of large Taq polymerase molecules.
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