Neutral Transfer of DNA Or RNA to Nitrocellulose

New England Bio Labs 1-3

Southern Blot Protocol Neutral Transfer of DNA or RNA to Nitrocellulose or Nytran SuPerCharge Nylon Membranes

1. Make Neutral Transfer Buffers (one liter each): 2. Denaturing Buffer: a. Combine the following 0.5 M NaOH 20g NaOH 1.5 M NaCl 87.66 g NaCl

b. Bring to 1L with Milli-Q H2O 3. 20X SSC: a. Combine the following 3 M NaCl 175.5 g NaCl

b. Bring to 1L with Milli-Q H2O 4. Transfer Buffer: a. Combine the following

0.3 Na citrate 88.2 g Na citrate (Na3C6H507 • 2H2O)

800 ml Milli-Q H2O b. Adjust the pH to 7.0 c. Bring to 1L with Milli-Q H2O 5. Neutralizing Buffer: a. Combine the following 0.5 M Tris-HCl pH 7.0 60.56 g Tris 1.5 M NaCl 87.66 g NaCl

800ml Milli-Q H2O b. Adjust the pH with concentrated HCl c. Bring to 1L with Milli-Q H2O 6. Denature DNA gels: a. Place gel in denaturing buffer for 30 minutes at room temperature. b. Shake the dish containing the gel slowly. c. Rinse the gel with distilled water and transfer to neutralizing buffer. d. Shake gel slowly for 30 minutes at room temperature. e. Soak gel in 20X SSC transfer buffer f. Shake gel for 30 minutes slowly. 7. Treat RNA gels: a. For RNA gels which have been run in 2.2 M formaldehyde: i. Rinse four times in deionized water ii. Maintain in low ionic strength conditions prior to transfer. 1. Milli-Q H2O 2. Low-salt sodium b. For RNA gels which have been run in 0.41 M formaldehyde: i. No gel washing is required prior to transfer. 8. Make the TurboBlotter RNase-free: a. Wash the TurboBlotter device in detergent. b. Rinse the device completely with Milli-Q H2O c. Dry with ethanol (EtOH) New England Bio Labs 2-3

d. Soak TurboBlotter device in 3% Hydrogen Peroxide (H2O2) for 10 minutes at room temperature. e. Rinse completely with Milli-Q H2O that has been treated with 0.1% DEPC. 9. Set up transfer from gel to membrane: a. Wet and immerse nitrocellulose or nylon membrane in distilled water. b. Soak membrane in 20X SSC transfer buffer for 5 minutes c. Place “stack tray” of transfer device on bench, making sure it is level. d. Place 20 sheets of dry GB004 blotting paper (thick) in stack tray. e. Place 4 sheets of dry GB002 blotting paper (thin) on top of stack. f. Soak one sheet of GB002 blotting paper in transfer buffer g. Place the one sheet of presoaked GB002 blotting paper on the stack. h. Place the transfer membrane on the stack. i. Cover the membrane with the agarose gel j. Cut the gel to the size of the membrane, making sure there are no air bubbles between the gel and the membrane. i. If the gel is smaller than the blotting paper used, cover excess blotting paper with plastic warp of Parafilm to prevent wicking of buffer through the paper rather than the gel. k. Wet the top surface of the gel with transfer buffer l. Soak three sheets of GB002 blotting paper in transfer buffer m. Place the 3 sheets of presoaked GB002 blotting paper on top of the gel. n. Attach the “buffer tray” of the transfer device to the bottom tray using the circular alignment buttons to align both trays. o. Fill the buffer tray with transfer buffer. i. 125 ml for 7 x 8 cm to 11 x 14 cm transfers ii. 200 ml for 12 x 21 cm to 20 x 25 cm transfers 10. Start the transfer 11. Soak the pre-cut “buffer wick” (included in each blotter stack) in transfer buffer. 12. Connect the gel stack with the buffer tray using the “buffer wick”. 13. Place the wick across the stack a. The short dimension of the wick must completely cover the blotting stack b. Both ends of the long dimmesion of the wick must extend into the buffer tray. c. Make sure the edges of the wick are immersed in the transfer buffer. 14. Place the “wick cover” on top of the stack to prevent evaporation. a. Do not place any other weight on top of the “wick cover” during transfer. b. This is unnecessary and may inhibit transfer by crashing the pore structure of the agarose gel. 15. Transfer for 3 hours. a. Additional transfer time may be required for gels thicker than 4 mm or for larger sized nucleic acids. 16. Neutralize the membrane: a. Gently wash the transfer membrane in 2X SSC for 5 minutes b. Dry the membrane 17. Immobilize the DNA or RNA: New England Bio Labs 3-3

a. Place the membrane on a fresh sheet of dry GB002 blotting paper to remove any excess of 2X SSC buffer. b. Bake membrane at 80˚C for 20 minutes to 2 hours. i. A vacuum oven is recommended for baking of nitrocellulose membranes. ii. Fixation of DNA or RNA requires only 20 minutes of baking, or until the blot has dried completely. iii. The more blots are placed in the oven, the more time will be required. c. As an alternative to drying on Nitrocellulose membranes, the DNA or RNA may also be covalently bound to the membrane by crosslinking the molecule to the matrix in the presence of UV light. i. Expose the blot to a source of UV light (254 nm) for a total dose of 120 mJ/cm2 for a damp membrane. 18. Store the blots at this point desiccated at 4˚C for several months. 19. Hybridize and detect using desired method. 20. Refer to the membrane instruction manuals for complete protocols.