Random Amplified Polymorphic DNA (RAPD)

Requirements
1. Thermal Cycler
2. Genomic DNA
3.Deoxynucleotide triphosphate (dNTPS-dGTP, dATP, dTTP, dCTP)
4.10X PCR buffer (MgCl, KCl, Tris)
5. Random Primer
6.TaqDNA Polymerase
7.Agarose and EtBr
8.Tris-Borate buffer
9.DNA Marker

Amplification performed with total reaction volume of 20μl
Label sterile 0.5ml eppendrof tube; add a drop of mineral oil.
Add the following PCR reagents one after the other; use fresh tips for each reagents

2mM dNTP Mix - 2μl
ddH20 - 7.7μl
10x assay buffer - 2μl
Random Primer - 3μl
Tad DNA polymerase - 0.5μl
Template DNA (5-40ng) - 5μl
Centrifuge at 2,000rpm for 30sec. (to mix all reagents).
Load in thermocycler.
Programming condition for Random Amplification is as follows:
CYCLING CONDITIONS
Cycle 5(1) initial denaturation at 95ºC 2 min
Cycle 6(35)
Step 1. 1min at 95ºC to denature the DNA
Step 2. 1sec at 36ºC to anneal the primers
Step 3. 2 min at 72ºC to extend the annealed the primers.
Cycle 7 (1) final extension step of 5 min at 72ºC-to ensure complete extension of amplified products.

Cycle 7 4ºC hold (optional)

Agarose Gel Electrophoresis

Sample preparation
8μl of PCR amplified products
5μl of loading dye

Seal both the sides of the gel-casting tray and place the clean comb in the respective position.
Weigh 1.5%agarose and add 1xTAE buffer
Boil till agarose dissolves completely (clear solution)
Add etbr to the melted agarose, mix well (1μl/10ml of agarose), cool it under running tapwater.
Pour the agaorse in the central portion of the casting tray; let it solidify at room temperature or in fridge.
Remove the seal of the casting tray and place the solidified gel in gel tank and pour required volume of 1xTAE till gel submerges in buffer, Gently lift the comb.
Connect the power cord to power supply.
(Red-anode, Black Cathode)
Load the samples in the well along with marker (10μl)
Run at 100V for 30mins.
Visualize the banding pattern under UV Transilluminator.