I want to ask that I have isolated DNA from bacterial samples but the problem is that i'm not getting any amplification using the same isolated DNA for PCR I want to give it a proteinase k Treatment again but I don't know the exact amount of proteinase k to be used for 25-30 microlitre of DNA
Hi Asghar,
Hope this will help you
https://www.promega.com/-/media/files/resources/protocols/product-information-sheets/n/proteinase-k-protocol.pdf
1 ul of 10 mg/mL proteinase K.
I don't think that the small amount of proteins hinder PCR and there may be other reasons. Even in colony PCR, amplification is observed with little amount of plasmid, in the presence of comparatively higher amount of proteins.
And after digestion of cells, denature the proteanase k with 85c for 30-45minutes, or it will chew up your taq in the pcr rxn.
Best
I agree the Second part of Mangal Singh's comment. It could be other reasons. Had you included a positive control in your PCR experiment? The positive control shoul give you a band in normal condition.
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