STH2063 Molecular Techniques in Ecology

RAPD

Random Amplified Polymorphic DNA (RAPD)

Laboratory protocol
1) Take out your DNA from freezer. Make sure the DNA is fully melt before proceed to step 2. 2. Prepare your PCR cocktail using the recipe below on ice:

Component miliQH2O (RO, sterile) 5X Reaction buffer dNTP mix (10 mM) MgCl2 (15 mM) Primer (10 M) Taq polymerase (5 units/L) Template DNA (10 ng/L) Total of Reaction

1X reaction 11.5 L 4.0 L 0.6 L 1.6 L 1.0 L 0.3 L 1.0 L 20.0 L

10X reactions 115 L 40.0 L 6.0 L 16.0 L 10.0 L 3.0 L 1.0 L

- Prepare your mastermix (e.g 10 X reactions) in a 1.5ml microfuge tube. - Label your PCR tube accordingly. - Pipette 19l of PCR mastermix into each PCR tube containing 1l of your DNA samples. - Put one drop of mineral oil in the PCR tube after each reaction has been added if necessary. 3. Run your PCR using the following profile in a thermocycler:

Step Denaturation Denaturation Annealing Extension Extension Soak

Temp. 94 oC 94 oC 43.9 oC 72 oC 72 oC 4 oC

Time 3 min 30 s 30 s 30 s 5 min

No. Cycles 1

35 1

4. Take out your PCR tube from the thermocycler, and keep in -20 C or directly use for electrophoresis. -Dont forget to turn off the thermocycler. 5. Run the PCR reaction in a 1.5% agarose gel (premixed with ethidium bromide) electrophoresis for 90 mins at 80V. 6. Visualized the gel under UV and take or save photograph accordingly. 9. Write a report on your experiment and submit to the lecturer the following week.