Isolation of Plasmid DNA

7 EXPERIMENT No: 4 DATE 31.10.2022 ISOLATION OF PLASMID DNA - Alkaline lyris method AIM small Male plasmid preparation from E cole MATERIALS REQUIRED LB broth Amphisillin 0.2 N NaOH Gumose Tris HCI EDTA 5M Sodium autate PH 4.8 Ethanol 1% SDS RNAse H Distilled water Eppendorf tubes untrifuge PROCEDURE 1. Transfer 2 mL E.cote culture to eppendent tube, centrifuge at 300 rpm for 1min. Remove supernatant 2. collect the pallet, add 100ml GTE (solution) keep It in ice for 2 min 3. immediately add 200ul of freshly prepared NaoH 4 SDS (solution() up gently tap the tubes 4. inwibate the tubes for 2min, add 150ml of sodium/potassium acet are solution (solution III) 41 mix gently 5. keep the tubes at room temp. for max 5 min and centufuge at 5000 rpm for 2min 6. Discard the pellet ul carefully transfer the supernatant to a fresh ependorf tube 7. Add 2 volumes of ice cold ethanotto the tube and incubate for 20 min at 20°C 8. After incubation centrifuge 14,000 rpm for 15 min 9. wash pellet In IML 70% etnanol up centrifuge at 5000 rpm for 5min 10 Dry the pellet at room temperature to remove etnamol

8 11. Re suspend the pullet in 30ml in Tris EDIA, gently tap 12. Add 111 RNAse H 13 Mix 10u1 sample + 2 ul bromophenol blue 14. Load samples in 0.8% agarose gel GEL CASTING prepare 30 mL 0.8% agarose solution using TBE buffer Seal the tray using cellophane Boil and dissolve agaross. Add 3 ul Et.Br. cool to 50-60°. Pour into get tray with comb. Allow It to solidify with TBE buffer Mix 10ul DNA + 2u1 gel loading dye Remove comband tape. Mount the gel in tank filled Load the sample into wells, electrophonesis at 50V OBSERVATION Orange colour DNA bands were observed, that indica tes the presence of plasmid RESULTS Plasmid DNA of E. coli was succesfully isolated so Kduizz