Scientific Calculations

You wish to store a sample in TE buffer which comprises of 5mM Tris-HCl and 1mM EDTA). You have a stock solution of 2M Tris-HCl. How much of the stock solution of Tris-HCl will you add to make 150mL of TE buffer?

Answer explanation

C1 = 2M (2000mM)

V1 = ?

C2 = 5mM

V2 = 150mL

C1V1 = C2V2

2000 x V2 = 5 x 150

V2 = 750 / 2000

V2 = 0.375 mL or 375uL

Calculate the amount of 10uM forward primer solution to add to a PCR reaction (25uL total reaction) to make a final concentration of 0.4uM forward primer in the reaction.

Answer explanation

V1=(0.4x25)/10, V1=10/10, V1=1

Calculate the amount of water you need to add to make a final concentration of 70% ethanol solution by using 100mL of pure (100%) ethanol.

Answer explanation

V2=(100x100)/70, V2=10 000/70, V2=142.9 – we already have 100mL pure ethanol so we need to add 42.9mL water to make it 70%

You have ordered some PCR primers. They have arrived as dried (lyophilised) pellets. You have 30nmol of your forward primer. What volume of water should you resuspend the primer solution in to give a final stock concentration of 100uM?

Answer explanation

Forward primer: n=10nmol = 10 x 10^-9 mol

Stock concentration: c = 100uM = 100 x 10^-6 mol/L

n = cv therefore v = n/c

v = 10 x 10^-9 mol / 100 x 10^-6 mol/L

v = 0.0003 L = 0.3mL = 300uL

You have been given a cell line (CELL LINE A) at a concentration of 5 x 10⁶/mL. You want to extract DNA from only 1.6 x 10⁶ cells. Calculate what volume of cells you need to use for this experiment.

Answer explanation

V1=(1.6 x 1)/5, V1=1.6/5, V1=0.320mL or 320uL

You’ve now extracted the DNA (CELL LINE A) and want to determine the concentration. First you want to perform a 1 in 5 dilution using water. You need 150uL final volume to run on the spectrophotometer. How much of your DNA sample will you use?

You run the diluted sample on the spectrophotometer and receive the following results (water blank has already been subtracted):

Sample: A₂₆₀= 0.315, A₂₈₀=0.180

Remembering that 1OD at A₂₆₀= 50ug/mL of double-stranded DNA, what is the concentration of your DNA sample (CELL LINE A)

Answer explanation

0.315 x 50 x 5 = 78.75ug/mL (or 78.75ng/uL)

You are now going to set up a PCR. But first you need to dilute your sample to 40ng/uL in a total volume of 30uL for the PCR. What volume of your DNA sample (CELL LINE A @ 78.75ng/uL) do you need to use to make this dilution?

You are now going to prepare your mastermix. In addition to your sample, you have an additional 2 DNA samples to test. You also plan to set up a negative control (containing water instead of DNA template). How many reactions will you prepare a mastermix for?

Each PCR will consist of a final volume of 25uL. What volume of 10X PCR Buffer will you add to your mastermix to ensure there is a final concentration of 1X?

Answer explanation

For an individual sample you would need 2.5uL 10X PCR Buffer. In 5X mastermix you will need 12.5uL of 10X PCR Buffer