Rapid Isolation of Yeast Genome

5 EXPERIMENT NO:3 DATE 11.10.2022 RAPID ISOLATION OF YEAST GENOMIC DNA (Bust in Grab mened AIM To isolate yeast genomic DNA MATERIALS REQUIRED YPD Medium Trilon X-100 SDS NaCl Tris-HCI EDTA Phenol uncoroform Ethanol TE Buffer untrifuge Agarose gel electrophoresis system Spendroff tubes PROCEDURE 1. 30°C Transfer 1.5ml of liquid culture of Yeast (20-24h grown at Remove supernatant in YPD into ependsoff tube. centrifuge at 15,000 rpm, 5min 2. Add 200fl lysi's buffer into the pellet 3. water Immerse bath tubes in dry ice ethanol for 2 min; transfer to 95°c 4. for I min to thaw quickly Vortex for 30 sec. Add 200,41 phenol chloroform (I:D) vortex for and 5. untrifuge at 15,000 rpm for 3min, room temperature Transfer upper aqueous phase to another eppendroff containing Inversion 400,11 in cold 100% ethanol Mix gently by 6 Inuubate at room temperature for 5 min Alternatively. precipitate at -20°cto increase yield 7. centrifuge at 15,000 rpm, 5min, mentemperature. Remove supernatant with Pasteur pipetle 8. Wash pellet with 0.5 m (500ML) 70% ethanol, spindown as deseri- bed in step7 9. Remove supernatant. Air dry the pellets at room temperature for 5 min

10 Resuspend in 25.50 It buffer (samples from plates should be resuspended in 10ml buffer) 11. Add 0.2541 RNase Run the yeast genomic DNA n agarose gel clutrophoresis GEL CASTING Seal the tray using cellophane Prepare 30 ml 0.8% agarose solution Et.Br. using TBE buffer cool to 50-60°C - Pour into gel tray with comb Allow It to Boil and dissolve agarose Add 3111 solidify Remove comb and tape Mount the gel in tank filled uit TBE buffer Mix 10ul DNA + 2MI gel loading dye Load the sample into wells, electrophonesis at 50V OBSERVATION Orange colour bands were observed RESULT 5 Genomic DNA of yeast was successfully isolated by Bust and Grab' method