C stands for concentration, not amount. So in your case C1 would be 4.8 microgr/100 microliters and C2 0.2 microgr/30µl. If you do the calculation you will get a volume of 4.2 µl to have 200 ng of DNA in your 30 µl reaction volume. But in my opinion that´s way too much for a pcr as too much DNA can inhibit the reaction. We usually have 50 ng of genomic DNA in a reaction volume of 50 µl and it works fine. Your original volume of 1.25 µl which means 60 ng of DNA, although calculated incorrectly, is far more suitable in my opinion.

Thank you very much for the reply.

I tried to achieve your numbers (volume 4.2 microl for 200 ng and 60 ng in 1.25 microl), in order to understand the philosophy of calculation.

This is how I did it:

my original concentration is 4.8 micrograms/100 microliters, and in 30 microliters (the volume of my reaction) it will be 1.44 micrograms - I used the simple triple rule to calculate the ratio.

And then, to obtain 60 ng:

V1

_{} - the volume I take, which is 1.25 microl;

C

_{1} - concentration of DNA in volume of 30 microl, which is 1.44 micrograms;

V

_{2} - volume of reaction, which is 30 microl;

C

_{1} - concentration of DNA in my reaction volume of 30 microl.

1.25 * 1.44 / 30 = 0.06 microgr =

**60 ng**.

And also, to obtain 4.2 microliters:

V

_{1} - the requested volume;

C

_{1} - concentration of DNA in volume of 30 microl, which is 1.44 micrograms;

V

_{2} - volume of reaction, which is 30 microl;

C

_{1} - concentration of DNA in my reaction volume of 30 microl, which is 0.2 microgr (200 ng).

30 * 0.2 / 1.44 =

**4.16 microliters**.

As you can see, I got your numbers, but do not think this is a correct way to do the calculations, or...?

It is a shame I do not know such basic things, but nobody teached me. I will appreciate if you explain me more about this.