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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
Volume (in μL)
Volume (in μL)
Required for a 6
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
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
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
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
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
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
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.