Question

After digesting the plasmid, the student decides to amplify the Evan1 gene from the new organism. After isolating the DNA, the student gathers all the stock reagents needed to setup the PCR from the lab. They find the following reagents:

10mM of dNTPs

10X Buffer with Magnesium

5U/ul of Taq Polymerase

10 uM Ev1 Fwd Primer

10 uM Ev1 Reverse Primer

The student notices that there will not be enough dNTPs to setup their PCR reaction. They find a 50mM stock of each, dATP, dGTP, dCTP and dGTP, in the freezer and decide to use those to make 100ul of their 10mM stock of their dNTPs before proceeding. How many ul of each dNTP would be needed to make this stock? How much water would they need to add?

Answer 1

Stocks of dNTPs are usually given as the concentration of each dNTP in the stock, so a 10mM stock would have 10mM of each dNTP and a 2.5mM stock for example would have 2.5mM of each dNTP.

Using the formula M1V1=M2V2

M1= 50mM, V1= ?

M2= 10mM, V2= 100ul

Substituting in the above formula: 50 x V1= 10 x 100

V1= 1000/50= 20ul

So we would need 20ul of each dNTP in a total volume of 100ul to get a 10mM stock. That is, each dNTP should have a concentration of 10mM.

So we would take 20ul of dATP + 20ul of dGTP + 20ul of dTTP + 20ul of dCTP + 20ul of water to make up the volume to 100ul.

In other words, you have a 50mM stock and you need a 10mM working stock, ie., 1/5 of the original concentration. So for 100ul you would take 1/5 of 100= 20ul of each dNTP and then make up the volume with nuclease-free water, which would be 20ul after adding 80ul of all 4 dNTPs.