Primers and templates - Primer and template concentrations (May/14/2005 )

OK, I know this is quite thick, but I have just started to use PCR for my MSc project. I am trying to detect the ORF 73 region of human herpesvirus 8, which is 1743 bp. I have extracted viral DNA from human plasma. My standard primer is 48.8nM and my reverse primer is 50.7nM. I am using Roche High Fidelity Master Mix for a 50uL PCR.

I am confused however as to how to work out exactly the concentrations/volumes of my primer/template?

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That's the dry amount of your primers. You can first added 488 ul and 507 ul TE buffer to your primers to make 100 uM stock solution. Then take 10 ul from the stock and add 90 ul water to make 10 uM working solution of primer. For each 10 ul PCR reaction, add 0.2 ul primer from the working solution. For a 50 ul PCR reaction, add 1.0 ul primer.

Why don't you scale down your PCR reaction from 50 ul to 10 ul so you can save lot amount of reagents.

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Agree with maccas. Create stock and working solution of each primer.
Stock, 100uM is pretty standard.

Working concentration is really dependent on volume of PCR reaction. If you plan on sticking with 50ul due to need of sensitivity or large amount of pcr product for multiple downstream applications. Then 25uM-50uM is a good working concentration. Adding 1 ul to each reaction will give you a 0.5uM-1.0uM final concentration.


50 ng of DNA is good starting point. Since you are extracting from plasma and don't have to worry about a large amount of genomic dna, you could get away with way less.

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