Biotechnology and Bioinformatics (1+1)

Method

1. Place the gel in a clean, baked glass baking tray.

2. Depurinate the DNA in the gel with 0.25M HCL for 8 min (until the Bromophenol blue turns yellow), with occasional shaking. Rinse 2-3 times with distilled water.

3. Denature the DNA twice by soaking the gel for 15 min in several vol. of denaturation solution, with constant gentle shaking. Again rinse the gel 2-3 times with distilled water.

4. Neutralize the DNA twice by soaking the gel for 15 min in several vol. of neutralization solution. Then finally soak the treated gel in transfer solution (20X SSC)

5. Connect the pump inlet on the front panel to the liquid trap and connect the trap to the base of the VacuGene XL blotting unit.

6. Pre-wet the nylon membrane as recommended (either in H₂O or 2X SSC) and place it on the porous screen (nitrocellulose membranes can be sensitive to the NaOH solution). Place the plastic mask on the membrane in such a way that it overlaps on each side of the membrane by approximately 5mm.

7. Fit the top frame and secure it by tightening the four locking clamps.

8. Starting with one of the gel edges, let it gradually slide onto the membrane. Avoid trapping air bubbles. Make sure that gel and mask overlap by atleast 2mm. Small cracks and the walls in the gel must be filled with melted agarose to get a good seal. (All leakages can be sealed with agarose preferably low melting point agarose).

9. Switch on the VacuGene XL pump. Stabilize the vacuum between 50 and 55 mbar.

The gel must always be covered with transfer solution when the vacuum is on.

10. Carry–on the transfer for approximately 90mins. Ensure that the gel remains immersed during this time. Remove the transfer solution.

11. Turn off the pump. Mark the wells. Remove the gel.

12. Wash the filter in 6X SSC, to eliminate any bit of adhering agarose. Air dry the filter for 30 min. Bake the filter at 80⁰C for 2 hours under vacuum (200 mbar).