Submarine Gel Electrophoresis

SUBMARINE GEL ELECTROPHORESIS Requirements : 1.Submarine gel system 2.Mini power pack 3.UV Transilluminator. 4.DNA samples, standard marker Solutions: 1. SB Buffer: To prepare 250 ml, weigh Sodium hydroxide Boric acid Dis.water make up to

- 0.1 9 - 2.0g - 250ml(pH 8.0)

2.6X gel loading buffer (40% sucrose, 0.025%BPB) To prepare 1 ml Sucrose - 0.040g BPB Dis.water make up to

- trace - 1 ml

3.Ethidium Bromide (5μg/ml) To prepare 1 ml stock solution Ethidium bromide - 5 mg Double dis .water up to - 1 ml 4. Agarose Agarose SB buffer

( 1 %) 0.1 9 10 ml

(2%) 0.2g 10ml

PROCEDURE:

1. Clean the gel platform and comb seal the open ends with cellophane tape. 2. Keep the Agarose solution (10ml) in a boiling water bath and dissolve the agarose. 3. Cool down the agarose solution temperature to 56C. 4. Add 1μl Ethidium bromide to the Agarose solution, mix gently and Pour on to the gel platform gently. 5. Place the comb in the notch, and allow the gel to set. 6. Place the gel platform with comb in the submarine unit. 7. Pour 165 ml of SB buffer 2-3 mm above the surface of the gel. 8. Add 2ul of 6x gel loading buffer to 10μl of sample. 9. Keep it in boiling water bath for 3 minutes. 10. Remove the comb slowly from the gel and inject samples slowly in to the wells. 11. Continue the run till the BPB tracking dye reaches the anodic end. 12. Observe the bands in a UV transilluminator.