Hi,
My query is regarding the amount of DNA in my sample using Picogreen assay kit.
I normally use a total reaction volume of 100 microlitre out of which 50 microlitre is 1:200 fold diluted Picogreen (as per manufacturer recommendation) and 50 microlitre is standard DNA or my sample.
For the standard, the final concentration expressed in ng/ml is as per the manufacturer after diluting with TE buffer.
I use a 5 microlitre of a sample (which has been pre-diluted already 10 times before being used) and obtain a DNA concentration of
e.g 800 ng/ml, the actual concentration of my sample would be
= (800 ng/1000 ml)*20*10 = 160 ng/ ml (5 microlitre of sample in 100 microlitre of reaction volume and 10 fold prior dilution of sample).
Please suggest as I got faint band for normal tissues following agarose (1.5%) gel electrophoresis using TBE buffer.
Best
Debashish
by my calculation if 5ul ( or any volume really) of 1:10 dilution gives 800ug/ul then you only multiply by the dilution to get the original concentration ( there is no factor of 20 involved) so the original concentration is 8000ng/1000ul or 8ng/ul not the 160 that you calculate
Dear Paul,
I agree with you, which is what we normally do for protein estimation. In this particular Picogreen assay, the concentration of the standard is prepared while diluting with the buffer, TE and Dye, Picogreen. For this reason, I think we need to consider the dilution of the sample in the reaction mixture.
Please refer to the link for the original calculation.
http://www.nature.com/protocolexchange/system/uploads/3551/original/Quant-iT_Picogreen_dsDNA.pdf?1423512049
Best
Debashish
thank you for that information Debashish. I will think some more on this but meanwhile I suggest that you assay a sample of known dsDNA concentration against your standard and then apply the dilution factor and see what concentration it converts to. I think even one of the points on the standard curve of lambda dna treated as an unknown would determine if the factor od 20 is necessary
Shouldn't the "(800 ng/1000 ml)" be "(800 ng/1000 ul)"? 1mL = 1000uL....
Also, it looks like the measured sample was diluted 1:10 twice.
1. You said it was "pre-diluted" 10 times.
2. You used 5uL to obtain the final 50uL
From what I can tell your concentration is 80ng/uL....
Paul Rutland's suggestion to "back check" was right on....
DearKirk
You are correct. It should be 1000 microlitre instead of 1000 ml. The sample was diluted in total100 times but in the final reaction mixture the volume of the Picogreen is also taken into cobsideration for calculation of standard as per the manufacturer. So, the final dilution becomes 200.
Thanks.
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