Question

The student knows that the final concentrations for the PCR reagents are as follows:

0.2mM of dNTPs

1X Buffer with Magnesium

0.025U/ul of Taq Polymerase

1 uM Ev1 Fwd Primer 1 uM Ev1Reverse Primer Water

The student proceeds to carry out their experiment. They account for one tube to be used to amplify the Evan1 gene, and the other for their control. They decide to setup a 50ul total volume per tube for this reaction. Given this information, calculate the individual volumes of each reagent that will be needed to create the 2X master mix (list each reagent with final volume to be added on a separate line).

Answer 1

C1V1 = C2V2

For one reaction tube,

PCR reaction volume = 50 uL

dNTP stock concentration = 10 mM

Required concentration = 0.2 mM

Required volume, V1 = (0.2 X 50)/10 = 1 uL

Buffer stock concentration = 10X

Required concentration = 1X

Required volume, V1 = (1 X 50)/10 = 5 uL

Taq stock concentration = 0.025 U/uL

For 50 uL, Required Taq volume = 0.025 X 50 = 1.25 uL

Primer stock concentration = 100 uM

Required concentration = 1 uM

Required volume, V1 = (1 X 50)/100 = 0.5 uL

Double these values for two PCR reaction tubes.

If the stock concentration change, modify the final volumes accordingly.