Method: Hybridization to Southern Blots

June 24, 1990

Srini Ramachandra

Principle:

To hybridize radioactively labeled DNA probes to Southern blots containing human DNA to detect Restriction Fragment Length Polymorphosims (RFLPs). This method can also be used for most other applications (e.g., slot blots, zoo blots, yeast YAC blots etc.).

2 days for hybridization, 3-5 days for X-ray film exposure

Procedure:

Day 1

Pre-hybridization:

1. Rinse blots in Postwash II solution (0.1X SSC, 0.2%SDS). Place a piece of nylon mesh between every two blots and place the blots in a thermal sealing plastic blot bag. [At least 20 blots (20cm x 20cm each) can be hybridized in a single bag].
2. Add 25 ml of pre-hyb solution for 1-2 blots; increase volume for more blots (or enough to allow blots to slide slightly inside; use ~150ml for 20 blots). Double seal the bags completely with T-bar sealer and incubate at 50 degrees C for at least 30 minutes before adding the labeled probe. Leave enough open space in the bags to cut open and add probe. (You can also let the pre-hybridization incubation go overnight).

Hybridization:

THE FOLLOWING STEPS SHOULD BE CARRIED OUT BEHIND A PLEXIGLASS SHIELD

Check the radioactive work area with the monitor before starting to work.

1. If the probe is known to be "single copy" do not add competitor human DNA. Otherwise add 250 µl of sonicated human DNA (2.5 mg/ml stock) to the labeled probe DNA, place the tubes in the metal holder and denature DNA by boiling both the probe DNA mix and the labeled lambda DNA (used to reveal molecular weight standards) for 10 minutes. If the probe does not have human DNA added, 5 minutes of boiling is sufficient to denature DNA.
2. Quick-cool the tubes in ice for ~2 minutes with the tubes still in the metal holder to prevent lids from popping open.
3. Add the probe DNA and lambda DNA to ~3ml of pre-hyb solution in a 5 or 15 ml snap-cap tube, cap tightly and mix by inverting the tube. (Refer to Primer Extension labeling protocol for counts to be added).
4. Cut open the pre-hyb bag at one corner, pour the probe and pre-hyb mix into the bag and reseal the bag (double sealing is good). Check for leaks by pushing the liquid towards the seals.
5. Pass the bag over a sharp edge (e.g., the edge of the table) to help squeeze the hybridization mix evenly between the blots (the more blots in the bag, the more vigorous the mixing should be). Incubate the bags at 50 degrees C on the platform shaker in the radioactive-labeled incubator, overnight (16-24 hours).
6. Clean up the radioactive work area thoroughly after you are done. Refer to the 32P lab safety sheet.

Day 2

Post-hybridization washes to remove non-specifically bound probe:

STEPS 1 THRU 3 BELOW SHOULD BE DONE BEHIND THE PLEXIGLASS SHIELD AND CONTAMINATED GLOVES SHOULD BE CHANGED FREQUENTLY

1. Cut open one corner of the bag behind the shield and pour the radioactive hyb mix carefully into the liquid radioactive waste container. If you are working with several bags at once pull the blots out first, drain the hyb mix into a large beaker and then transfer the liquid waste to the liquid waste container.
2. Remove the blots from the bag and place in approximately 200 ml (or more according to the number of blots/bags, enough to immerse blots) of Postwash I solution [2X SSC (for 1 liter, mix 100 ml of 20X SSC with 900 ml of dH2O)] in a deep sided steel pan. The first wash solution should be disposed of in the liquid waste container. [The other washes can be poured down the sink marked for radioactive work. Record the amount of waste poured down the sink (a 25ng reaction is approximately equal to 0.05 µCi)].
3. Transfer the blots to a fresh 200 ml (or more) of Postwash I solution. Rub the blots individually at each wash, flip them over and rub again. Wash at room temperature in 2X SSC for a total of 30 minutes.

THE FOLLOWING STEPS CAN BE DONE OUTSIDE THE RADIOACTIVE WORK BENCH. Check the wash solution with the monitor and use your judgement.

1. Wash the blots twice for 30-45 minutes each wash at 65 degrees C in Postwash II solution [ 0.1x SSC, 0.2% SDS (for 500 ml, add 2.5ml 20X SSC and 10 ml 10% SDS to 487.5ml dH2O)] in a covered steel pan with gentle shaking. These washes should be done in the 65 degrees C shaker-baths meant for radioactive work only.

X-ray film exposures:

1. Take the blots out of the shaker-bath and press the blots between two pieces of Whatman 3MM paper to remove excess wash solution. Check the Whatman papers with a monitor ; if radioactivity is detected, wash blots one more time. DO NOT ALLOW THE BLOTS TO BECOME COMPLETELY DRY.
2. Place the semi-dry blots in blot bags (14" x 17") as follows: cut open the bag on one side so that two contiguous sides are open and lightly crumple the bags with your hands to remove static between the sheets of the blot bags. This will help in slipping the blots easily between the plastic sheets of the bags. Smooth the bags to get good contact between the blot and the bag. Place blots as close together as possible without overlap and make sure they are placed asymmetrically in the bag for orientation purposes. Try to accomodate as many blots as possible per bag.
3. Open the film holder (cardboard Kodak X-ray film exposure holder), place the blot bag inside, position the intensifying screen on top of the blot bag with the dull side of the screen in contact with blot bag. If multiple holders are used, orient all with the same side up as only one side of the holder has a lead lining.
4. In the darkroom (with the safelight ON) open the Kodak X-ray film box, remove a sheet of film (the film feels stiff between two sheets of paper) and place film between the intensifying screen and the blot bag. Close the cassette holder and close the clasp. The X-ray film box is kept in the cabinets in the processor room. It should always be stored in the dark, with the lid securely taped.
5. Place the film holder/s between the plexiglass or metal holder plates and store the holders in the -80 degrees C freezer for 3-5 days. The exposure time varies with the probes and the counts obtained. Prepare the film labels (printed labels); store them until the films are developed.
6. Remove the film holder/s from the freezer and thaw at room temperature for 1 to 2 hours before processing the films. This is to prevent the screens from cracking (if bent), due to the intense cold.
7. Develop the films using the Kodak X-ray film processor. Refer to the processor instructions written by Matt Holt in this manual.
8. Place the labels on the developed films; mark the individuals of the family or pedigree appropriately with Sharpee markers (Other markers may not be removable with ethanol). Mark the alleles also, if they are known.
9. If the film is overexposed, place another film in the holder and re-expose without the screen for 1 day. If the film is underexposed, re-expose with the intensifying screen for several days (up to 10 days maximum).

Stripping blots for reuse:

1. Remove blots (should be semi-moist) from the bags and place them in a steel pan containing appropriate amount of 0.1N NaOH. (For 500ml, add 5ml 10N NaOH to 495 ml dH2O). Shake the blots at room temperature for 30 minutes (no longer than 45 minutes) on a platform shaker. This removes the probe from the blot. Drain the wash.
2. Neutralize the blots with 0.2M Tris-HCl pH 8.0, 0.1X SSC, 0.5% SDS (for 500 ml, add 100 ml of 1M Tris-HCl pH8.0, 2.5 ml 20X SSC and 25 ml 10% SDS to 372.5 ml of dH2O). Shake the blots at room temperature for 30 minutes or more on a platform shaker.
3. Store the blots wet in sealed blot bags at room temperature or in the cold (4 degrees C) until further use. If the blots are in the general lab supply, sign them back into the record books, and file them in the appropriate places.

Solutions:

• Tris-mix

  for 1 liter	final concentration
  500 ml 1M Tris-HCl, pH 7.5	0.5 M Tris-HCl
  50 ml 0.5M EDTA, pH 8.0	25 mM EDTA
  50 ml 50 mg/ml Heparin	2.5 mg/ml Heparin
  150 ml 31/3 % Sodium pyrophosphate	0.5% Sodium pyrophosphate
  250 ml 10% Sarkosyl	2.5% Sarkosyl

• Pre-hyb solution

  for 1 liter	final concentration
  200 ml Tris-mix* (see above)	0.1M Tris-HCl
  200 ml 50% Dextran sulfate	10% Dextran sulfate
  200 ml 5M NaCl	1M NaCl
  300 ml Formamide	30% Formamide
  100 ml ddH2O

  Store at 4 degrees C.

• 50% Dextran sulfate
  For 200 ml: Add 100 g dextran sulfate to 130 ml of ddH2O in a 500 ml bottle, let it sit overnight to dissolve and store at room temperature. Wear a face mask while weighing out dextran sulfate.

• Heparin (50mg/ml)
  Dissolve 5 g heparin in 50 ml of ddH2O, adjust volume to 100 ml with ddH2O, filter sterilize and store at 4 degrees C.

• 10% Sarkosyl
  Dissolve 100 g N-lauroylsarcosine in 500 ml ddH2O and adjust volume to 1000 ml with dd2O. Store at room temperature.
• 5M Sodium chloride (NaCl)
  Dissolve 1168.8 g NaCl in 3000 ml of ddH2O, adjust volume to 4000ml with ddH2O in a 4 L carboy and store at room temperature.
• 10% SDS
  Dissolve 100 g sodium dodecyl sulfate (SDS) in 500 ml of ddH2O, adjust volume to 1000 ml and store at room temperature. Wear a face mask while weighing out SDS.
• 20X SSC (20 liters)
  Dissolve 3504 g NaCl and 1760 g sodium citrate in 10 L ddH2O. (Use a 20 L carboy.) Adjust the volume to 20 L with ddH2O, and the pH to 7.4 with several drops of concentrated HCl.

References:

E.M. Southern. (1975) "Detection of specific sequences among DNA fragments separated by gel electrophoresis." J. Mol. Biol. 98: 503.

Sambrook, J., Fritsch, E.F., and T. Maniatis. (1989) Molecular Cloning, A Laboratory Manual. Second edition. Cold Spring Harbor Laboratory Press. pp 9.47 - 9.58.