SOUTHERN BLOT HYBRIDIZATIONS

Prehybridization treatment of blots (genomic DNA) :
Insert filter(s) in heat seal bag and wet with 65°C hybridization buffer. Approximately 10 ml of hybridization solution is used for one to a few filters per bag. Increase volume slightly to adequately cover additional filters. The smaller the volume, the faster the rate of hybridization. Seal the bag, and reheat it to 65°C in an oven or water bath. Denature salmon sperm DNA at 95°C for 5 minutes, inject this into bag (25g needle), and reseal. There should be very few air bub bles in the bag. Prehybridize in a gently rotating (about 70 rpm), 65°C, water bath for several hours. Replace the solution with fresh hybridization buffer, add heat-denatured probe with another aliquot of salmon sperm DNA, and reseal. Hybridize overnight while gently rotating at 65°C.

Cloned DNA:
Cloned DNA blots may be prehybridized as above, but usually background is low enough to skip this step. Place filters in a bag, add 65°C hybridization solution, heat the ba g to 65°C in an oven or water bath and inject heat denatured probe. Hybridize while gently rotating at 65°C overnight.

Post hybridization filter treatment (genomic DNA): Cut the corner of bag and pour the hybridization solution into the radioactive aqueous waste container. Rinse the bag with 2X SSPE, 0.1% SDS, 1 mM EDTA (approximately 10 ml total volume). Add this to radioactive waste container. Open the bag and remove filter(s). Wash them in 200-500 ml of 65°C 2X SSP E, 0.1% SDS, 1 mM EDTA while shaking at approximately 70 rpm for 30 minutes. Transfer this wash solution to the radioactive waste container, and repeat this wash once. The solution from the remaining washes contains very little radioactivity. Dispose of it in accordance with your local standards. Wash twice with agitation for 30 minute per wash in 0.2X SSPE, 0.1% SDS, and 1 mM EDTA at 65°C. It is advisable to flip these filters over at 15 minute intervals.

Post Hybridization filte r treatment (Cloned DNA):
Rinse the opened bag with 65°C 5X SSPE, open the bag and wash 45 minutes, 65°C in 5X SSPE. Dispose of this solution in a radioactive aqueous waste container. Wash twice with 2X SSPE, followed by two washes in 1X SSPE, each for 30 minutes at room temperature. It is advisable to flip these filters over at 15 minute intervals.

Following these washing procedures, blots are air dried on a Whatman 3MM paper behind a Lucite shield. Tape the corners of the blots to a Whatman filter paper, labeled with 14C India ink or with fluorescent ink, and wrap in Saran wrap.

Autoradiography:
Optimal exposure times must be empirically determined, but the following intervals are offered as a guide when radiation is measured on a Ludlum model 3 survey meter with a 44-3 detector where 1 mR/hour is approximately 2500 counts per minute. For genomic DNA, cpm are usually at background. Expose a minimum of 4 days.
For cloned DNA, use the following chart to estimate exposure times:

CPM just above membrane Exposure Time
< 0.2 K Overnight
0.5 K 3-5 hours
1 K 30 minutes
3 K 20 minutes

In the dark room, place a piece of X-ray film on top of an intensifying screen, then place the Whatman filter containing the hybridized filter on top of the X-ray film with the nylon or nitrocellulose filter towards the film. Close the film cass ette, wrap in foil, label and expose in a -70°C freezer.

Removing Old Probes from Membranes for Rehybridization: Soak the membranes in 0.4 M NaOH at 42°C for 30 minutes with agitation. Transfer the membranes to 0.2 M Tris-HCl, pH 7.5; 0.5% SDS and soak for another 30 minutes at 42°C with agitation. Use autoradiography to check for efficient removal of the probe.

RECIPES

2X SSPE 0.1% SDS, 1mm EDTA
100 ml of 20X SSPE
10 ml of 10 % SDS
2 ml of 0.5 M EDTA
QS to 1 liter with distilled water; skip autoclaving.

0.2X SSPE 0.1% SDS, 1mm EDTA
10 ml of 20X SSPE
10 ml of 10% SDS
2 ml of 0.5M EDTA
QS to 1 liter with distilled water.

Hybridization Solution for cloned DNA
5x SSPE
10x Denhardt's Solution
0.1% SDS
Make in autoclaved water. Do not autoclave.

Hybridization Solution for Genomic DNA (30 ml)
7.5 ml 20x SSPE
1.5 ml 100x Denhardt's Solution
3 ml 50% dextran sulfate
1.5 ml 1M phosphate buffer, pH 6.7
0.3 ml 10% SDS
16.05 ml H2O
Add 150 µl of 10 mg/ml denatured sal mon sperm DNA for pre-hybridization.

100x Denhart's Solution (2% BSA, 2% Ficol, 2% PVP in 3x SSPE)
For 100 ml:
2% (w/v) BSA (Sigma Fraction V)
2% (w/v) Ficol (400,000 mw)
2% (w/v) Polyvinylpyrrolidone (40,000 mw)
Dissolve in 3x SSPE (diluted with autoclaved water) and filter sterilize. This solution may be stored at room temperature, 4° C or -30° C.
20X SSPE (3 M NaCl, 0.2 M NaH2PO4, 20 mM EDTA, pH 7.0)
350.7 g NaCl
55.2 g NaH2PO4(H20) (--or-- 48 g anhydrous NaH2PO4, mw = 120.0)
14.89 g EDTA
Dissolve in boiling water, cool to room temperature, & adjust pH to 7.0 with NaOH. QS to 2 liters with water an d autoclave.
Dehybridization of Nylon Membranes
Soak the membranes in 300 ml autoclaved 0.4 N NaOH, 42°C for 30 minutes with agitation. Then, soak the membranes in 0.1x SSPE, 0.2 M Tris-HCl, pH 7.5 and 0.5% SDS 42°C 30 minutes with agitation.

0.1xSSPE, 0.2M Tris-HCl, 0.5% SDS (500 ml)
2.5 ml 20xSSPE
25 ml 10% SDS
100 ml 1M Tris-HCl, pH 7.5
372.5 ml water

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Copyrig ht 1993, 1996, and 1997 by Mark Barton Frank, Ph.D.
Proper citation for data acquired from this document is: "Frank, M. B. Southern Blot Hybridizations. In: Frank, M. B. ed. Molecular Biology Protocols. (http://omrf.ouhsc.edu/~frank/dnahybrd.html). 1997. Oklahoma City. Revision Date: October 2, 1997."