Question

Question 5 Using stock solutions in a protocol: from volume to final concentration A DNA ligation reaction is carried out in a 25 μL reaction mix. This is a reaction carried out in cloning. You are going to do a number of these reactions so you decide to make up a larger volume of the reaction mixture, called a master mix. This saves on multiple repetitive pipetting and reduces errors. You have been given a protocol to make up 1 mL of this ligation master mix and it states that you must add 2 μL of a 100 mM ATP. What is the final concentration of ATP in the ligation reaction? You need to do 30 ligation reactions. Do you have enough reaction mixture with the 1 mL?

Answer 1

Concentration of 2 μl of 100 mM ATP in 1 ml (1000 μl) can be calculated using dilution formula m1v1 = m2v2. Where, m1 and m2 are the initial and final concentration, repectively; v1 and v2 are initial and final volumes. Therefore,

m1 = 100 mM

v1 = 2 μl

m2 = ? (we have to calculate)

v2 = 1000 μl

So, m2 = (2 x 100)/1000

           = 0.2 mM = 200 μM

The final concentration of ATP in the final ligation mix is 200 μM.

For one ligation reaction 25 μl is needed; therefore, for 30 reaction we will need = 25 x 30 μl

                                                                                                                  = 750 μl

Therefore, 1 ml or 1000 μl will be sufficient for 30 ligation reactions.