Question

Suppose you’re going to do 9 PCR reactions. The starting and final concentrations of the ingredients are listed below. Each reaction should have a volume of 50 µl.

Component, stock concentration Final Concentration or amount
5X Reaction Buffer 1.0X
MgCl2, 25 mM 1.5 mM
Nucleotide Mix, 20mM 0.2 mM
Primer 1, 10 µM 1.0 µM
Primer 2, 10 µM 1.0 µM
Taq DNA Polymerase, 5 Units/µl 1.0 Units
Template DNA, concentration unknown use 1 µl per reaction
(a) Briefly explain how you would set up the reactions and show all your calculations. Indicate the amounts that you would pipet into your tubes. Be clear. (Show ALL your calculations on a piece of paper, take a picture and paste it in the space below).
(b) Using the amounts listed above, how many pmol of each primer will there be in each reaction? (Show ALL your calculations on a piece of paper, take a picture and paste it in the space below).

Answer 1

To prepare the 9 PCR reactions, volumes of each component are calculated based on the desired final concentration for a 50 µl reaction volume.

Step-by-step explanation:

To set up 9 PCR reactions with the final volume of 50 µl, we first need to calculate the volume of each component required per reaction based on the final concentration and then multiply by 9 for all reactions.

5X Reaction Buffer: To get to 1.0X from a 5X stock, you need 1/5th of the final volume which is 10 µl per reaction. So for 9 reactions, it's 90 µl.

MgCl2: To get 1.5 mM from 25 mM, the dilution factor is 25/1.5. So we need 50 µl/ (25/1.5) = 3 µl per reaction. For 9 reactions, it's 27 µl.

Nucleotide Mix: To get 0.2 mM from 20 mM, the dilution factor is 20/0.2. So we need 50 µl / (20/0.2) = 0.5 µl per reaction. For 9 reactions, that's 4.5 µl.

Primers: For 1 µM from a 10 µM stock, use 5 µl. So, 5 µl each of Primer 1 and Primer 2, which results in a total of 90 µl for each primer over all reactions.

Taq DNA Polymerase: For 1 Unit from 5 Units/µl, we need 0.2 µl per reaction, resulting in 1.8 µl for 9 reactions.

1 µl of Template DNA is added directly to each reaction.

The rest of the volume to 50 µl will be made up with distilled water for each PCR reaction.

Quantity of Primers in pmol

To calculate the number of pmol of each primer in each reaction: since 1 µl of 1 µM primer solution contains 1 pmol of primer, we will use 1 µl of each primer, so we will have 1 pmol of each primer in each reaction.