( Genomic DNA EXTRACTION (ANIMAL TISSUE) | INTRODUCTION The Medox-Bio DNA purification kit isolates genomic DNA (gDNA) from animal tissues. The phenol-chloroform method is the most frequently used for DNA isolation, which removes proteins and other cellular components. The yield of DNA is quantitative and reproducible, and the DNAis of sufficient purity for direct use in any application, e.g. PCR, restriction digestion, cloning, DNA sequencing, Southern blot analysis, etc. PRINCIPLE DNA in the animal tissues is released by using lysis buffer. The cell membrane of the animal tissue is weakened by EDTA and SDS denatures the cellular proteins in the solution |. Extraction with phenol denatures the protein, Chloroform removes SDS- Protein/polysaccharide complexes as white interface between the aqueous and organic phase after centrifugation and isoamyl alcohol prevents foaming. The high molecular weight genomic DNA in aqueous phase is precipitated with 100 % ethanol or isopropanol (solution-Il) and further the salts are removed by washing with 70% alcohol.MATERIALS REQUIRED BUT NOT PROVIDED 1. Incubator 2. Electrophoresis apparatus with power pack 3. UV Transilluminator 4. Micropipette and tips PROCEDURE All the micropipettes should be well calibrated and the pipetting should be accurate to get optimum results. 4. To the 0.1g of animal tissue, add 0.8ml of Solution-I and homogenize well until the tissue completely homogenize well into a slurry. 2. Add 10ul of Proteinase-K; gently tap the tube to mix well. Leave it for 30 minutes at 65°C.a 10. 11 12. Add 0.5 ml of solution-Il, mix well and centrifuge at 8000 rpm for 5 minutes at 4°C Take the supernatant (aqueous phase), add 500ul of Solution-lll, mix well and centrifuge at 7000 rpm for 5 minutes at 4°C. Transfer the aqueous phase to a fresh tube. Add double the volumes (1ml) of ice-cold solution-IV and add 100ul of solution V and mix gently to precipitate the DNAand incubate at -20°C for 15 minutes. Centrifuge at 10000 rpm for 10 minutes at 4°C and discard the supernatant. Wash the DNA pellet once with 500u! of 70 % Ethanol and give a short spin to remove alcohol. Dry the pellet properly and ensure that whole ethanol is dried off. Dissolve the pellet in 25yl of TE buffer, add 5ul RNase and incubate at 37°C for 30 min. Store the DNAsample at -20°C until further use. Check purity and average fragment size by Agarose gel electrophoresis. PREPARATION OF 0.8% AGAROSE GEL AND ELECTROPHORESIS Me Prepare 30ml of 0.8% Agarose solution in the buffer provided (0.5X TEB) comb.10. 1 12. 13. 14. Boil until the agarose is completely dissolved and no obvious particle of agarose remain in the suspension. When the gel temperature is around 40°C, add 4yl of Ethidium bromide. Mix properly. Seal the gel-casting tray on two sides and place the comb on the tray in appropriate place. Pour the agarose mixture in to the tray-containing Allow the agarose to solidify in the tray. Then remove the seal from the two sides of the tray without disturbing the gel. Then keep the gel tray in tank containing 0.5X TEB buffer with the wells in the cathode (negative) side. The buffer level in the tank should be maintained above the gel tray. Gently lift the comb to avoid damage of the wells, the gel is now ready for loading. Connect the cords between the electrophoresis tank and the power pack before loading the samples To prepare sample for electrophoresis, mix 20u! of sample DNAwith 5ul of gel loading dye provided. Load the samples into a respective wells along with 5yl of DNA Marker. After loading, switch on the power pack and adjust the voltage to 50V or 100V. Continue the electrophoresis until the dye reaches to1/3rd ofthe above. Observe the bands under UV Transilluminator.RESULT AND INTERPRETATION Genomic DNA isolated from animal tissue is observed as a single band on (0.8%) Agarose gel. 1 2 40000bp g000bp 6000bp 5000bp 4000bp 3000bp 2000bp 1500p 1000bp 500bp LANE 1: 1Kb DNA Ladder LANE 2: Liver Tissue DNA From Bovine