Biotechnology and Bioinformatics (1+1)

Procedure

1. Prepare 1% gel using ultrapure agarose

(0.5 g of agarose in 25 ml of TBE /TAE buffer)

TBE Buffer (10x - 1 lit)

Tris - 108 g

Boric acid - 55 g

EDTA - 9.3 g

pH - 8.3

To dissolve the agarose boil for few min in microwave oven.

2. After cooling add 1 m l of Ethidium Bromide and swirl gently without bubbling.

(EtBr stock - 10 mg/ml, 5 m l /100 ml of buffer or agarose mix)

3. Pour the gel gently in the gel tray after placing comb and leave for 30 min.

4. Fill the gel tank with TBE buffer.

5. Place the gel tray in the tank after removing the combs from the gel gently. Lift the combs without shaking from both sides.

6. Mix 1 m l of loading buffer with 2 m l of sample DNA and loaded in the wells. Use separate tips for each sample to avoid cross-contamination.

7. Load marker DNA also.

8. Run the gel at 160 V (80 milli amperes) for a period of 45 min. (Before loading the samples the apparatus can be checked by connecting to electric circuit. On both sides from the electrodes bubbles will come. This ensures the powerpack is in good condition)

9. Stop the gel run when the tracking dye migrated the other end.

10. Observe the gel in the UV Transilluminator using goggles.

Note : Take care while handling the gel because it contains ethidium bromide a carcinogen. Always use gloves. Discard the TBE buffer.