Question

The table below shows the stock (i.e. what you have) and final (i.e. what you want) concentrations/quantities for a 25 μl PCR.

Item	Stock Concentration	Amount wanted per 25μl PCR
Forward Primer	10 μM	0.4 μM
Reverse Primer	10 μM	0.4 μM
Buffer (contains dNTPs, polymerase, MgCl2)	2X concentrate	1X
DNA	25 ng/μl	25 ng/μl

*Use the table to answer the following questions and please show how you got the answer. Thank you!

1. For each 25 μl reaction, what volume of each primer is required?

2. For each 25 μl reaction, what volume of buffer is required?

3. For each 25 μl reaction, what volume of DNA is required?

4. For each 25 μl reaction, what volume of water is required?

5. You have 6 DNA samples. You plan to make a PCR master mix for a total of 8 reactions (6 DNA samples + 1 negative control + 1 extra reaction to account for pipetting error). What will be the volume of each ingredient that you use to make your PCR master mix (forward primer; reverse primer; buffer; water)?

Answer 1

Answer 1

For 25ul reaction,

Volume of forward primer required is

We applied M1V1=M2V2 formula

where M1 (Initial concentration) = 10uM

V1 (Initial volume) = ?

M2 (Final concentration)= 0.4 uM

V2 (Final volume)= 25 ul

10 X V1 = 0.4 X 25

V1 = 1ul

Volume of reverse primer required is

We applied M1V1=M2V2 formula

where M1 (Initial concentration) = 10uM

V1 (Initial volume) = ?

M2 (Final concentration)= 0.4 uM

V2 (Final volume)= 25 ul

10 X V1 = 0.4 X 25

V1 = 1ul

Answer 2

Volume of Buffer required is

We applied M1V1=M2V2 formula

where M1 (Initial concentration) = 2X

V1 (Initial volume) = ?

M2 (Final concentration)= 1X

V2 (Final volume)= 25 ul

2 X V1 = 1 X 25

V1 = 12.5 ul

Answer 3

Volume of DNA required is

Concentration of DNA is 25ng/ul

Required concentration 25 ng per PCR reaction

So, 1 ul

we need only 1 ul

Answer 4

Volume of water required

So, Water volume - 12.5 ul (buffer)- 1 DNA- 1 ul (forward primer)- 1ul (reverse primrer) = 25 (total volume)

Volume of water = 9.5ul

Answer 5

For 8 reactions =

Buffer required = 12.5 ul (per reaction) so for 8 reactions = 100ul

Forward primer = 1 ul (per reaction) so for 8 reactions = 8 ul

Reverse primer = 1 ul (per reaction) so for 8 reactions = 8 ul

water = 9.5 ul per reaction so , 9.5 X 8 = 76 ul