RAPD

Coconut (Cocos nucifera L.)

Random Amplified Polymorphic DNA (RAPD)

The Random Amplified Polymorphic DNA (RAPD) method is based on the Polymerase Chain Reaction (PCR) using short (usually 10 nucleotide) primers of arbitrary sequences. Polymorphism of amplified fragments are caused by: (1) base substitutions or deletions in the priming sites, (2) Insertions that render priming sites too distant to support amplification, or (3) insertions or deletions that change the size of the amplified fragment.

Materials

Equipment: Thermocycler,Power supply Unit
Water: Sterile de-ionized or distilled water should be used for preparing all the reagents and pre-mixes
Reaction buffers: Assay buffer for Taq DNA polymerase ( supplied by the manufacturer of Taq DNA polymerase)
Deoxynucleoside triphosphates (dNTP'S): 2.5 mM each of dCTP,dATP,dTTP,dGTP. Readymade solutions of dNTPS are available from many manufactures. Store at –20 ºC
Magnesium chloride: 25mM stock and store at –20 º C
Taq DNA polymerase
Genomic DNA 5-25 ng/ml stocks. DNA of sufficient quality can be obtained in coconut by using SDS protocol.

Methods

Assemble RAPD reactions as follows:

2.5µl

DNA stock (25ng/µl)

2.5µl

Assay buffer containing 2.5mM MgCl2 (2.5mM)

1µl

MgCl2 stock (1.5mM)

1µl

primer stock (25pmol)

4µl

dNTPS (400µM)

1µl

Taq polymerase (1U)

Sterile water to make 25 µl

Wear gloves throughout RAPD reaction preparation procedure. Assay buffer, dNTPs, MgCl2 and primer solution are thawed from frozen stock. Keep the assembled reaction in themocycler for amplification.

Amplify DNA in themocycler: Cycling conditions may be modified depending on the thermocycer used

Temperature profile

General cycling steps followed are:

Step1: Initial denaturation at 94º C for 5.00 min
Step2: Denaturation at 94º C for 1.00min
Step3: Primer annealing at 55º C for 1.00min
Step4: Primer extension at 72º C for 2.00min
Step5: Go to 2, 39 times
Step6: Final extension at 72º C for 10min
Step7: 4º C for ever

After the reaction, DNA is analyzed through gel electrophoresis.

Agarose gel electrophoresis

Reagents for agarose gel electrophoresis

Agarose, TBE/TAE buffer, ethidium bromide*, gel loading dye,

To prepare 100ml of a 0.7% agarose solution, measure 0.7g agarose into a glass beaker or flask and add 100ml 1X TBE or TAE.

Microwave or stir on a hot plate until agarose is dissolved and solution is clear.

Allow solution to cool to about 55º C before pouring. ( ethidium bromide can be added at this point to concentration of 0.5µg/ml

Place the comb in gel tray.

Pour 50º C gel solution into tray to a depth of about 5mm. Allow the gel to solidify for about 20 min at room temperature.

To run, gently remove the comb, place the tray in electrophoresis chamber, and cover (just until wells are submerged) with electrophoresis buffer (the same buffer used to prepare the agarose).

To the RAPD sample from refrigerator, add 1µl of 6%gel loading dye for every 5 µl of DNA solution. Mix well. Load 20µl of DNA per well. Load also the DNA size standards along side RAPD reactions.

Connect the electrodes to the power pack. and electrophoresis at 50-150Volts until the bromophenol blue dye has reached three fourth of the gel length.

Stain the gel with ethidium bromide (if not already included in the gel).

Examine the gel under UV light (transilluminator).

Depending on the objective of the experiment make a note of polymorphism, segregating bands, and appearance of overall pattern with in fingerprint.

Bands may be sized by comparison to molecular weight standards. The standards should be used to generate a standard curve for interpolation.

After you have run the gel, obtain a photograph, and label and measure the migration of the DNA bands. Make a standard curve plot of the known size markers, and determine the size of the marker bands.

Gel Interpretation

Bands are sized and matched directly on gels, or photographic films, or photocopies on transparency overlays.

Note the presence and absence of bands.

Analyze the data using computer software NTSYS / RAPDistance

Applications of RAPD

RAPD protocol can be used in genetic mapping and finger printing application in other palms like arecanut and oilpalm also.

Note: Ethidium bromide is a mutagen and a probable carcinogen. Wear gloves when working with ethidium bromide solutions. Also use care not to contaminate the work area with the solution. UV light is damaging and must be used with caution. UV light causes burns and can damage the eyes.

Coconut (Cocos nucifera L.)
Random Amplified Polymorphic DNA (RAPD)

The Random Amplified Polymorphic DNA (RAPD) method is based on the Polymerase Chain Reaction (PCR) using short (usually 10 nucleotide) primers of arbitrary sequences. Polymorphism of amplified fragments are caused by: (1) base substitutions or deletions in the priming sites, (2) Insertions that render priming sites too distant to support amplification, or (3) insertions or deletions that change the size of the amplified fragment.
Materials
Equipment: Thermocycler,Power supply Unit Water: Sterile de-ionized or distilled water should be used for preparing all the reagents and pre-mixes Reaction buffers: Assay buffer for Taq DNA polymerase ( supplied by the manufacturer of Taq DNA polymerase) Deoxynucleoside triphosphates (dNTP'S): 2.5 mM each of dCTP,dATP,dTTP,dGTP. Readymade solutions of dNTPS are available from many manufactures. Store at –20 ºC Magnesium chloride: 25mM stock and store at –20 º C Taq DNA polymerase Genomic DNA 5-25 ng/ml stocks. DNA of sufficient quality can be obtained in coconut by using SDS protocol.
Methods
Assemble RAPD reactions as follows:
	2.5µl	DNA stock (25ng/µl)
	2.5µl	Assay buffer containing 2.5mM MgCl2 (2.5mM)
	1µl	MgCl2 stock (1.5mM)
	1µl	primer stock (25pmol)
	4µl	dNTPS (400µM)
	1µl	Taq polymerase (1U)
	Sterile water to make 25 µl
Wear gloves throughout RAPD reaction preparation procedure. Assay buffer, dNTPs, MgCl2 and primer solution are thawed from frozen stock. Keep the assembled reaction in themocycler for amplification. Amplify DNA in themocycler: Cycling conditions may be modified depending on the thermocycer used
Temperature profile
General cycling steps followed are: Step1: Initial denaturation at 94º C for 5.00 min Step2: Denaturation at 94º C for 1.00min Step3: Primer annealing at 55º C for 1.00min Step4: Primer extension at 72º C for 2.00min Step5: Go to 2, 39 times Step6: Final extension at 72º C for 10min Step7: 4º C for ever After the reaction, DNA is analyzed through gel electrophoresis.
Agarose gel electrophoresis
Reagents for agarose gel electrophoresis
Agarose, TBE/TAE buffer, ethidium bromide*, gel loading dye, To prepare 100ml of a 0.7% agarose solution, measure 0.7g agarose into a glass beaker or flask and add 100ml 1X TBE or TAE. Microwave or stir on a hot plate until agarose is dissolved and solution is clear. Allow solution to cool to about 55º C before pouring. ( ethidium bromide can be added at this point to concentration of 0.5µg/ml Place the comb in gel tray. Pour 50º C gel solution into tray to a depth of about 5mm. Allow the gel to solidify for about 20 min at room temperature. To run, gently remove the comb, place the tray in electrophoresis chamber, and cover (just until wells are submerged) with electrophoresis buffer (the same buffer used to prepare the agarose). To the RAPD sample from refrigerator, add 1µl of 6%gel loading dye for every 5 µl of DNA solution. Mix well. Load 20µl of DNA per well. Load also the DNA size standards along side RAPD reactions. Connect the electrodes to the power pack. and electrophoresis at 50-150Volts until the bromophenol blue dye has reached three fourth of the gel length. Stain the gel with ethidium bromide (if not already included in the gel). Examine the gel under UV light (transilluminator). Depending on the objective of the experiment make a note of polymorphism, segregating bands, and appearance of overall pattern with in fingerprint. Bands may be sized by comparison to molecular weight standards. The standards should be used to generate a standard curve for interpolation. After you have run the gel, obtain a photograph, and label and measure the migration of the DNA bands. Make a standard curve plot of the known size markers, and determine the size of the marker bands.
Gel Interpretation
Bands are sized and matched directly on gels, or photographic films, or photocopies on transparency overlays. Note the presence and absence of bands. Analyze the data using computer software NTSYS / RAPDistance
Applications of RAPD
RAPD protocol can be used in genetic mapping and finger printing application in other palms like arecanut and oilpalm also.

Note: Ethidium bromide is a mutagen and a probable carcinogen. Wear gloves when working with ethidium bromide solutions. Also use care not to contaminate the work area with the solution. UV light is damaging and must be used with caution. UV light causes burns and can damage the eyes.