1. Run 1 - 5 µg gDNA per lane, photograph the gel with a fluorescent ruler.
2. Soak gel in 0.25 M HCl for 15 minutes (hydrolyzes long molecules into shorter ones for transfer).
3. Rinse briefly in water.
4. Soak gel in denaturing buffer 2 X 30 min. at RT with gentle aggitation.
5. Soak gel in transfer buffer for 15 min with gentle aggitation. At the same time, wet and soak the Nytran membrane in dH 2 O.
6. Place the bottom half of the TurboBlotting tray on a level bench (see diagram on next page).
7. Place 20 sheets of dry absorbent paper (or appropriately sized paper towels) inside the bottom half of the blotting tray.
8. Place 4 sheets of dry blotting paper (or 3MM paper) on top of the stack.
9. Place one sheet of wet (soaked in transfer buffer) blotting paper on top of the stack.
10. Place the prewetted Nytran on top of the stack.
11. Place the gel on top of the Nytra. Surround the gel with parafilm if necessary. Make sure there are no air bubbles between the gel and the Nytran.
12. Wet the surface of the gel with a small amount (about 1 ml) of transfer buffer and place 3 sheets of wet (transfer buffer) blotting paper on top of the gel.
13. Attach the top half of the buffer tray to the bottom half. Add 125 ml of transfer
    buffer to the tray.
14. Start the transfer by connecting the gel stack to the buffer using a wick that has been prewetted in transfer buffer. Make sure the entire gel is covered with wick.
15. Place a wick cover on top to prevent evaporation.
16. Continue transfer for 1-4 hours.
17. After the transfer is complete, remove the Nytran and gel as a single unit. Mark the membrane with a VWR lab marker to indicate which side has the DNA. Also, cut the gel and membrane along the bottom to the wells and notch the top left corner of the Nytran to give it an orientation.
18. Gently wash the membrane in 100 ml of 1X neutralizing buffer for 5 min. (Use 5X stock solution)
19. Place the Nytran, DNA side up, on a clean paper towel and allow it to air dry for about 5 minutes. Then, cross-link the DNA to the Nytran with 2.5 minutes of UV irradiation. The blot is ready for prehybridization.

Special Notes

• Do not allow RNA to be transferred O/N; weak signal due to hydrolyzed RNA.
• O/N transfer of DNA to Nytran is OK but not to nitrocellulose.
• Membranes can be dried for 15 min at 80 C but only Nytran can be dried at RT.
• To strip a blot: 3 X 5 min. at 80° C in 0.5% SDS; 40 mM Tris-HCl, pH 7.8.
• 0.25 M HCl = 5.1 ml conc. HCl in 250 ml total volume

Reagents for Alkaline TuroBlotting

Nytran

• Schleicher & Schuell membrane (0.2 µm pore size)
• 7 x 10 cm S&S # 77797

Denaturing Buffer:

• 175.5 g = 3 M NaCl
• 16.0 g = 0.4 M NaOH
• 873 ml dH2O

Transfer Buffer: (pH can be neutralized by CO2 - store air tight)

• 175.5 g = 3 M NaCl
• 0.32 g = 8 mM NaOH
• 0.63 g = 2 mM Sarkosyl
• 879 ml dH2O

5X Neutralizing Buffer:
1.0 M phosphate buffer, pH 6.8

• 79.5 g Na2HPO4
• 52.39 g anhydrous NaH2PO4
• (or 60.25 g NaH2PO4*H2O)
• bring to 1 L with water

* indicates prewet in transfer buffer

Lab Schedule Outlined

Lab Schedule In Context of Research Project