Hi
Let's take as example the following situation.
We want to prepare a 50 ul PCR product. Below there are the stock concentrations and the concentrations we need.
DNA polymerase: 5000 units/ml --> 1.25 units
Buffer: 10x --> 1x
dNTP mix: 2.5 mM each --> 200 uM
Primer 1: 10 uM --> 10 pmol
Primer 2: 25 uM --> 10 pmol
Template: 785 ug/ml --> 500 ng
Water: up to 50 ul
This is an exercise I got in a Molecular Biology class where the solution of the problem was like that:
DNA polymerase: 1.25 units / 5000 units/ml = 0.00025 ml = 0.25 ul
Buffer: 1 / 10 * 50 ul = 5 ul
dNTP mix: 2.5 mM each => dATP = 2.5 mM; dTTP = 2.5 mM; dGTP = 2.5 mM; dCTP = 2.5 mM => 2.5 mM * 4 (dATP + dTTP + dGTP + dCTP) = 10 mM = 10000 uM
C1V1 = C2V2 => V1 = C2V2 / C1 => V1 = 50 ul * 200 uM / 10000 uM = 1 ul
Primer 1:
10 uM/ml = 10 pmol/ul => 10 uM/10 uM = 1 ul
Primer 2: 10 uM/25 uM = 0.4 ul
Template:
ug/ml = ng/ul => 500 ug / 785 ug/ml = 0.63 ul
Water: 50 ul - (0.25 ul + 5 ul + 1 ul + 1 ul + 0.4 ul + 0.63 ul) = 41.72 ul
I don't understand two things:
1. Why in some cases we care about the final volume and in others we don't? For example, the dNTP mix is expressed in mM and we count it according to the final volume. On the other hand, the primers are also expressed in uM but we don't care about the final volume.
2. Why 10 uM/ml = 10 pmol/ul when 1 uM = 1000000 pmol but 1 ml = 1000 ul?
Can someone help me? Please!!!