Biochemical Techniques and Instrumentation

Procedure

1. Digest 10μg high molecular weight DNA in a total volume of 45μl with 50-100 units of restriction enzyme and 1/10th volume of the appropriate 10X RE buffer for 4h overnight at 37°C. Add 1μl of RNase for the last 1h of digest
2. Pour a 0.8-1% agarose gel in TAE buffer with ethidium bromide (10 mg/ml) using a comb containing 20 mm x 1 cm wells.
3. Add 1/10 volume (5μl) of 10X DNA loading buffer to each sample.
4. Load gel and include 15μl of premixed size markers (lambda HindIII/uX174 HaeIII) in both of the outside lanes.
5. Run the gel at 120 V for 5-6h at room temperature. Recirculate buffers at 3h.
6. Photograph the gel with a ruler next to the size markers and cut off the upper left hand corner of the gel. Soak the gel for 1h with gentle shaking in 300-500 ml of denaturation buffer at room temperature.
7. Pour off denaturation solution and rinse the gel with a small amount of renaturation solution.
8. Soak the gel for 2 x 30 min at room temperature in 500 ml of renaturation buffer with gentle shaking.
9. Put on disposable gloves and set up a standard blotting chamber using 10X SSC as the transfer buffer
10. Cut a piece of 0.45 micron nitrocellulose membrane to exactly the size of the gel. Keep hands off of nitrocellulose. Drop the nitrocellulose onto the surface of a glass dish containing doubly distilled water and wet completely. Then submerge the nitrocellulose in the water.
11. Flip the gel over (so bottom of gel is facing up) and place onto the Whatman paper on the bottom of the blotting apparatus. Note: Place 4 sheets of Whatman on top of the blotting stone.
12. Using a spatula surround the gel with pieces of parafilm which underlap the gel by 1 mm on each side.
13. Transfer the nitrocellulose to a dish containing 10X SSC for 30-60 sec.
14. Place the nitrocellulose on top of the gel and roll out any bubbles with a piece of disposable 5 ml pipet. Cut 2 pieces of Whatman 3 MM paper to exactly the size of the gel and quickly soak in 10X SSC before placing on top of the gel. Roll out bubbles with the 5 ml pipet.
15. Cut a stack of paper towels to the size of the gel and place on top of the Whatman strips.
16. Place the top on the blotting apparatus and weight down with a 0.5 kg weight or a 500 ml bottle filled with water.
17. Blot overnight.
18. Put on gloves. Remove and discard paper towels and Whatman filters. Carefully remove the nitrocellulose sheet with the gel in position and flip over onto a clean glass plate. Mark the wells with a pencil and remove and discard the gel. Cut upper left hand corner of nitrocellulose.
19. Soak the nitrocellulose briefly (1-2 min) in 2X SSC.
20. Place nitrocellulose face up on a piece of dry Whatman paper and air dry 15-20 min.
21. Place the nitrocellulose between 2 pieces of precut Whatman paper and tape shut into a folder. Bake for 2h at 80°C in a vacuum oven.