MATERIALS AND METHODS

Sample collection
The catfish Clarias dussumieri were collected from different natural water bodies in Kerala. Clarias collected from water bodies of Ankamali,Kizhakkekotta,Kodungalloor,Moovattupuzha and Aluva (Eranakulam) and Rajakumari(Idukki). Fresh specimens were spot examined for specific morphological characters that define the catfish. The tissue samples from each of the specimens were collected aseptically and preserved in 90% ethanol.

Requirements
1) Refigerated centrifuge 2) Water bath 3) Micropipettes with disposable tips 4) 1.5ml autoclaved eppendroff tubes 5) Reagents

Reagents
Solution 1 Tris buffer (1M) - 500 l EDTA (0.5M) - 400l SDS - 2 ml Solution 2 Saturated NaCl solution (6M)

DNA extraction 20 mg of fish tissue samples taken in 1.5ml sterilized eppendroff

tubes and add 500 l of solution 1. Homogenize tissue samples with sterile homogeniser . Add 5l of Proteinase K (20mg/ml) from freezer. Quick vortex. Incubate at 55C in water bath for 2 hours with occasional mixing. Can stop here by leaving sample at 37C overnight but may need to add more proteinase K. After 2 hours incubation samples chill on ice for 10 minutes. Add 250l solution 2 (saturated NaCl) and invert several times to mix. Chill on ice for for 5 minutes. Spin at 8000rpm for 15 minutes. Carefully collect 400l clear supernatant into a new labeled eppendroff tube. Add 1000l of 100% ethanol to precipitate the DNA. Kept at -20C overnight.. After overnight incubation , samples spin at 11000 rpm for 15 minutes. Remove the supernatant. Rinse DNA pellet in 500l of cold 70% ethanol. Spin at 11000 rpm for 5 minutes. Carefully remove supernatant , pipette off excess liquid and partially dry the pellet at room temperature for 3 to 4 hours. Resuspend partially dried DNA samples in 70l sterile distilled water . Run about 5l of DNA samples in 0.8% agarose gel to check the presence of DNA. Store the remaining DNA samples at -20C.

RAPD In order to randomly amplify the gene sequences of the Clarias DNA, RAPD reactions were conducted using the primers OPK4,OPK-7,POK-8,OPK14,OPU-5 and OPK-4 . The amplification reaction were performed in a total volume of 25l. The reaction mixture contains ; Master mix - 12.5l Primer - 1.5l Template - 1l Milliq water - 10l The reactions were conducted using a thermal cycler under the following conditions ; Initial denaturation - 95C , 5 minutes Denaturation - 94C , 30 seconds Primer extension - 40C , 1 minute and 30 seconds Final extension - 72C , 10 minutes Total number of cycle is 35 To ensure that the reactions yielded adequate amplicon sizes, RAPD products were electrophoresed and visualized on 1.5% agarose gels containing ethidium bromide.

Agarose gel preparation

Agarose 0.4g 1X TBE buffer 5ml Distilled buffer 45ml Ethidium bromide 3l Heat agarose solution. Add 1.5l of ethidum bromide before cooling. Pour it in gel casting plate with already adjusted gel comb. Let it solidify at room temperature for the gel to set , before loading gel. Running agarose gel using cold 1X TBE buffer. Observed under UV.