Hello,
I have a template pDNA with concentration of 200 ng/uL and I need to add around 4ng in a total of 25 uL of PCR solution. How many uL do I need to add from the stock template DNA?
Do I need to dilute that and then?
Many thanks for your help. I'm going crazy
Hi Federica Casolari ,
Given [initial concentration: 200ng/µl] x [initial volume: x?] = [final concentration: 4ng/µl] x [final volume: 25µl]
So, x=(4 x 25)/200 , x= 0.5µl ==> 0.5 of DNA added to 24.5 supermix or 1µl of DNA to 49µl of supermix recation.
A protocol calling for "4 ng" and "a final concentration of 4 ng/µl" are two different things (4 ng vs 100 ng). I would double-check the wording in the protocol.
If you need to add 4 ng total, the easiest way would be to dilute your stock by adding 1 µl of stock pDNA to 49 µl of water to make a 4 ng/µl solution. Then you can add 1 µl of this diluted pDNA to your PCR reaction. Generally you should avoid pipetting volumes less than 1 µl to ensure accuracy of the measurement.
Hi, using the formula: C1*V1=C2*V2, you can use 0.5 uL in the final reaction.
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