Question

Question 23:                                                                                                  (10 Marks)

a) Given the stock concentration and desired final amount, or desired final concentration, of the following PCR components, calculate the volume of each you would add to a 25 μl PCR reaction mixture.

b) You want to analyse the resultant PCR product by agarose electrophoresis. Calculate how much agarose you need to use to make up 50 ml of 1.5 % gel. How much of 5x concentrated loading buffer will you add to load the whole sample on the gel?

Component	[stock]	[final]
oligonucleotide 1	10 μM	12.5 pmoles
oligonucleotide 2	5 μM	12.5 pmoles
Phusion polymerase HF buffer	5x	1x
dNTP mixture	10 mM each	5 nmoles each
MgCl2	25mM	0.5mM
template DNA	1 μg/ml	100 pg
Phusion polymerase	2 U/ μl	0.5 U
H2O

Answer 1

One by one:

Oligonucleotide 1:

Oligonucleotide 2:

Buffer:

We need to have a 5-fold dilution, this means that the added volume has to be 1/5 of the final volume, that is 5 microliters.

dNTP:

MgCl₂: We use the dilution formula:

Template DNA:

Phusion polymerase:

If we add all of these volumes, we get a total of:

10.1 microliters. This means that the amount of water will be:

b)

If you want to prepare 1.5% agarose gel (50 mL), you need to have 1.5 g of gel in 100 mL of gel solutions. If half of this volume is needed (50 mL), then half of the amount of gel will be needed, that is: 0.75 g.

If the loading buffer has to be diluted by 5, its volume has to be 1/5 of the total final volume (the sample volume of 25 uL and the buffer volume). This is:

Which can be rearranged and solved as: