Southern blot hybridization

Blot DNA from gel to membrane

  1. Run DNA samples in 1% agarose gel
  2. Rinse gel with distilled water
  3. Soak gel in 0.25 M HCl for 30 min
  4. Rinse gel with distilled water
  5. Transfer gel to 0.2 N NaOH for 30 min
  6. Prepare transfer chamber using glass tray and plastic plate. Place plastic plate on top of grass tray as if forming a bridge across the tray. This will serve as the transfer chamber.
  7. Add 0.2 N NaOH into the transfer chamber.
  8. Place Whatman paper on top of the plastic plate and immerse two sides of Whatman paper into 0.2 N NaOH solution to serve as a bridge. Be sure the Whatman paper is long enough to have both ends immersed in the NaOH solution.
  9. Soak Whatman paper with 0.2 N NaOH and get rid of all the bubbles using 10 ml pipette to roll over the paper on plastic plate
  10. Use glass plate to pick gel and put it face down on top of the wet Whatman paper.
  11. Cut membrane to the same size as gel and soak it in 0.2 N NaOH. Then place it on top of the gel.
  12. Get rid of all air bubbles using 10 ml pipette to roll over the gel and membrane.
  13. Cut 4 pieces of Whatman papers to the same size of gel
  14. Soak two Whatman papers in 0.2 N NaOH and place them on top of the membrane
  15. Place two dry Whatman papers on top of the wet Whatman papers.
  16. Place a stack of white paper towels on top of filter papers and use a book to place weight onto the whole stack. Let stand overnight.
  17. Take off all wet paper towels and remove membrane. Soak in 2X SSC for 15 min
  18. Let membrane dry on Whatman paper or paper towel.
  19. Membrane is ready to be used.

Pre-hybridization of membrane

  1. Pre-heat water bath and the rotating incubator in hot room at 65°C
  2. Place hybridization buffer in 65°C water bath for 15 min
  3. Place a membrane in hybridization tube
  4. Add 65°C hybridization buffer into hybridization tube (30 ml for big tube and 15 ml for small tube)
  5. Boil water in the lab and boil ssDNA stock 5 mg/ml for 5 min then place on ice immediately for 5 min
  6. Add ssDNA (stock 5 mg/ml) to the hybridization tube for the final concentration of 100 ug/ml
  7. Place hybridization tube with membrane in 65°C rotating incubator, and incubate overnight for new membrane or 2 hours for previously hybridized membrane

Radioactive labeling

  1. Prepare probe with 50 ng DNA and 40 ul distilled water in a microfuge tube
  2. Boil water and place probe in boiling water for 5 min then immediately place on ice for 5 min
  3. Transfer probe into RediPrimeII (Random prime labeling system) labeling tube. Use pipette to help dissolve the labeling mixture in labeling tube.
  4. Move to Hot room and start working under the hood

“After this step need to work carefully for radioactive material”

  1. Add 3 – 5 ul of P32 dCTP into RediprimeII labeling tube and incubate at room temperature under the hood for 2 hours (3 ul for new radioactive and 5 ul for old radioactive)
  2. Boil water in hood in Hot room and place RediprimeII labeling tube in boiling water for 5 min, then immediately place on ice with water for 5 min
  3. Bring hybridization tube with membrane to the hood and open the lid
  4. Transfer labeled radioactive probe into the hybridization tube and tighten lid
  5. Put hybridization tube back into 65°C rotating incubator. Don’t forget to balance the rotor with the same size hybridization tube
  6. Incubate in rotating incubator at 65°C overnight

Wash membrane after hybridization

“Work carefully for radioactive material”

  1. Pre heat water bath in Hot room to 65°C and place washing buffers, listed below, in the water bath 15 –20 min before start washing the membrane

2 X SSC in 0.1% SDS

1 X SSC in 01% SDS

0.5 X SSC in 0.1% SDS

0.1 X SSC in 0.1% SDS

  1. Discard hybridization buffer in special waste container for liquid radioactive materials in locked room
  2. Add 50 – 100 ml of 65°C 2 x SSC in 0.1% SDS and incubate in 65°C rotate incubator for 15 min
  3. Discard washing buffer to regular sink in hot room with running water, keep water running for 5 min after discarding the buffer
  4. Take membrane out from hybridization tube and check radioactive signal. If the signal from the membrane is between 10-20 cpm (count per minute), the membrane is ready to be dried under hood. If not, put membrane back into the hybridization tube and keep washing.
  5. Repeat step 3 to 5 with 1 X SSC in 0.1% SDS, 0.5 X SSC in 0.1% SDS, and 0.1 X SSC in 0.1% SDS respectively until the membrane has signal between 10 – 20 cpm.
  6. Let membrane dry under hood in hot room for 5 – 10 min
  7. Wrap membrane with plastic wrap
  8. Place membrane in exposure cassette
  9. Bring exposure cassette with membrane and X-ray film box into the dark room
  10. Turn off the lights in dark room, then open the X-ray film box
  11. Take one X-ray film out and place it on top of the membrane in the exposure cassette then slide the locks to close the exposure cassette
  12. Close the X-ray film box completely. Then turn lights on in the dark room
  13. Place cassette set in -80°C freezer for 2-3 days

Develop X-ray film

  1. Turn on the developing machine in dark room and push “run” button on the front of the machine. Machine will need to warm up for 15 to 20 min before it is ready for use. (Both red lights are on when the machine is ready)
  2. Remove exposure cassette with X-ray film from –80°C freezer and bring to the dark room.
  3. Turn off the lights in dark room. Then open the exposure cassette.
  4. Remove X-ray film from the cassette and place it into the right side of the developing machine. The X-ray film will be fed into the machine. When finished, the machine will beep. This means the machine is ready for the next X-ray film. Continue feeding in X-ray film until there is no more X-ray film to be developed.
  5. All developed film will come out on the left side on top on the machine.
  6. Turn light back on after finish developing all film
  7. Turn off machine by switching the red button on the right side from “on” to “off”.
  8. Bring the exposure cassettes to the hood and open them to dry for 10 min
  9. The same membrane can be re-used for hybridization with different probes by striping the old probe and storing membrane in -20°C in hot room next to the hood.

Striping old probe from membrane

  1. Soak used membrane in 0.2 N NaOH on shaker for 20 min
  2. Transfer membrane into 0.2 M Tris-HCl (pH 7.4) + 0.1% SDS and soak for 20 min
  3. Take membrane out and check radioactive signal

If we can still detect signal from the membrane, repeat step 1 to 3.

  1. Until we can not detect signal, soak the membrane in 2 X SSC for 5 min
  2. Let membrane air dry under the hood in Hot room for 5 min
  3. Membrane is ready to be used again.

Hybridization buffer

1 M Na2HPO4 (pH 7.2, FW = 140) 350 ml (autoclaved)

1 M NaH2PO4 (pH 7.2, FW = 120)150 ml (autoclaved)

0.5 M EDTA Na (pH 8.0, FW = 372.2) 2 ml (autoclaved)

20% SDS (Lauryl Sulfate)350 ml

Adjust the pH to 7.2 using: NaOH or HCl

Adjust volume to 1 liter with ddH2O