OPTIMISATION OF PCR AMPLIFICATION REACTION CONDITIONS

1. In a sterile PCR tube, set up the following master mix for 6 reactions (as shown in the

shaded column in the table below).

Use a new pipette tip for each reagent addition in order to prevent cross contamination

of stock reagents.

PCR Reagent Final

Concentration

Volume (in μL)

required per

reaction

Volume (in μL)

Required for a 6

Reaction Master

Mix

10X PCR buffer 1X 5 30

dNTPs (10 mM stock) 0.16 mM 4 24

MgCl2 (25 mM stock) 1.5 mM 3 18

Taq DNA polymerase 1U 0.2 1.2

Sterile H2O - 27.8 166.8

Final volume - 40 240

2. Dilute the template DNA (150 ng/uL) 10 fold to produce the required concentration for

PCR.

To do this, place 9 uL of sterile H2O into a microcentrifuge tube and add to this 1 uL of

the stock DNA template.

Mix well by pipetting up and down.

3. Using the master mix prepared in step 1 above, set up the following 5 PCR reactions,

which will each contain different concentrations of the forward and reverse primers.

Make sure to use the diluted template DNA prepared in step 2 above.

The volume of each PCR reaction will be 50 μL. 

91335 Molecular Biology 2 Subject Manual Autumn 2016

Page 89

Tube 1 Tube 2 Tube 3 Tube 4 Tube 5

Reagent (0.1 µM) (0.2 µM) (0.3 µM) (0.4 µM) (0.8 µM)

Template DNA (15 ng/µl) 1 μL 1 μL 1 μL 1 μL 1 μL

Master mix 40 μL 40 μL 40 μL 40 μL 40 μL

Forward primer (10 µM) 0.5 μL 1.0 μL 1.5 μL 2 μL 4 μL

Reverse Primer (10µM) 0.5 μL 1.0 μL 1.5 μL 2 μL 4 μL

Sterile H2O 8 μL 7 μL 6 μL 5 μL 1 μL

Final volume 50 μL 50 μL 50 μL 50 μL 50 μL

4. Vortex tubes to mix and then spin briefly to ensure all the contents are at the bottom of

the tube.

5. Place tubes into the ice bucket provided on the issues bench. Please make sure ALL

TUBES have been labelled with your initials.

6. The tubes will be placed into a PCR machine and exposed to the following amplification

parameters:

95°C x 60 seconds X 1 cycle

95°C x 20 seconds

65°C x 20 seconds X 34 cycles

72°C x 20 seconds

4°C hold

7. While the PCR in progress, the demonstrators will select some student volunteers

to prepare the agarose gels (containing gel red) for the class.

Instructions for the preparation of 1% (w/v) agarose gels will be provided.

8. Upon completion of the PCR, 15 µL of each reaction will be electrophoresed for

analysis.

8 To 6 fresh microcentrifuge tubes (labelled 1-5 and Std), add 5 µL of loading dye.

9. From the completed PCR reactions, remove 15 µL from each of the reactions (1-5) and

place sequentially into tubes 1-5 containing the loading dye. 



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