Biotechnology and Bioinformatics (1+1)

Procedure

1. Wash the plates neatly with distilled water and dry

2. Wipe the plates with 0.1% SDS solution and clean them properly

3. Fix the strips with vacuum grease and seal them with 1% agar solution(Check for leakage with distilled water)

4. Prepare the solutions as described above and pour the gel. Overlay the gel with 0.15 SDS solution, care should be taken to note that the upper layer is even and gel is 2 cm below the notch.

5. Allow the gel to polymerise uniformly

6. After proper polymerization remove the overlay solution and wipe it with filter paper

7. Then place the comb and pour stacking gel

8. Allow stacking gel to polymerise. Polymerization must be even and must extended uniformly up between fingers.

9. Sample preparation:

Prepare the samples as per the respective procedure and estimate the amount of protein present. For coomassie blue staining, load 150μg protein per lot. Take care that the volume should be minimum (20-40μl)

10. Take the precast gel and remove the base strip and wipe the extra grease with the help of cotton

11. Set up tank, connect leads and fix the gel. Pour running buffer in the lower chamber

12. Boil the sample with equal volume of sample buffer in a boiling water bath for 2 minutes

13. Load the sample with the help of a micro pipette or micro syringe. Start loading from one end of the gel. Don’t load the outermost wells because the tracks may be distorted. Fill the unused wells with sample buffer

14. Fill the upper reservoir with running buffer and overlay the samples with the same

15. With the help of a syringe and bent needle remove the bubbles under the gel between the glass plates

16. Run the gel at 20mA constant current until bromophenol blue reaches the bottom (6-8hrs)

17. Care should be taken to check that the plates do not get heated

18. After completion, open the plates and remove the gel

19. Wash the gel to remove SDS and stain it for 3h

20. Then destain till the background becomes clear

CAUTION

1. Pour the gel, preferably 24h prior to running

2. Take care that the sample volume and the amount of protein in the sample should be equal in all slots

3. While loading the sample, work as quickly as possible. If a well is empty and the adjacent well is filled, sample will leak through the finger into the empty well and cross contaminate

4. Don’t overfill the wells. Overfilling leads to cross lane contamination

5. When filling the wells fill it slowly with the upper reservoir buffer without disturbing the sample