Southern Hybridization – Molecular Cytogenetics Group Protocol – September 2009 – www.methods.molcyt.com

Link to downloadable Southern Hybridization Word document. Link to old protocol (has more about labelling and detection and some altneratives)

Agarose electrophoresis – run gels (0.8% to 1.3% typically depending of fragment sizes to be separated) and photograph after staining with ethidium bromide. Ensure a ruler is photographed next to the gel. Avoid the marker lanes being symmetrical (to help with later orientation).

Southern transfer

• Put gel into 0.25M HCl (5 times the volume of the gel) and shake gently at room temperature for 15 min (this depurinates and fragments the DNA so it binds to the membrane; blue marker changes to yellow colour; remove as soon as colour changes).
• Denature gel in 0.5M NaOH + 1.5 M NaCl (5 times gel volume) and shake gently at room temperature for 30 min.
• Neutralize gel in 0.5M Tris-HCl pH 7.4 + 1.5 M NaCl (5x gel volume) and shake gently at room temperature for 30 min.
• Set up gel (upside down) on bridge over a piece of Whatman 3MM filter paper soaked in 10x SSC, roll out any bubbles, put charged nylon membrane (soaked in 10x SSC; eg Amersham Hybond N+) on top, put clingfilm or parafilm around edge, place two gel-sized sheets of 3MM paper cut to gel-size on top, then put 10cm stack of paper towels on top, followed by 500g weight, eg Sigma catalogue). Fill area around bridge with at least 10x gel volume of 10xSSC.
• Leave for 10x SSC to be drawn through gel into towels overnight.
• Remove membrane and soak in 6x SSC for 2 min, blot dry and bake for 2hr at 80°C.
• Membrane can then be wrapped in cling film and stored at 4°C.

Depurination: (1000 ml) 0.25M HCl: 21.6 ml of 11.6M HCl in 1litre water (care – gets hot).
Denaturation: (1000 ml): 87.7g NaCL (1.5M) + 20g NaOH (0.5M) (care – gets warm).
Neutralization : (1000 ml): 87.7g NaCl (1.5M) + 500 ml of 1M Tris-HCl pH7.4
(All three can be autoclaved for long-term storage at room temperature).
Mark membrane at edge with pencil identifier, possible also cutting off a corner to help alignment.

Southern Hybridization
Stock solutions

• 20x SSC 1000ml NaCl 3M 175.3 g + NaCitrate 0.3M (88.2 g) autoclave
• 10% SDS
• 50x Denhardt’ solution   - (Ficol (type 400) 500mg; PVP powder 500 mg; BSA 500mg; Water to 50ml) - Aliquot to 10 ml and store at -20C
• 50% dextran sulfate
• 20% SDS

Prehybridization
Use 5 ml per 100 cm^2 of membrane

Rehydrate membrane in 4xSSC+0.5% SDS. Put into roller bottle with prehybridization solution and rotate for 4hr at 55 °C. Pour off solution and can be stored at -20C for reuse.

Hybridization

                               
Hybridize overnight at 55°C in roller bottle.
Post-hybridization and detection
Pour out probe mix and keep at -20°C for possible re-use.
Rinse membrane twice in 20 ml 5x SSC/0.1% SDS at c. 55 °C then for 10 min twice in 75 ml at 55°C.
Give stringent wash in 3x SSC/0.1% SDS at exactly 55°C. (Use 2x SSC for higher stringency).

 For probe labelling:

 Use RadPrime DNA Labeling System (Invitrogen) 

1. Denature 25 ng (1 µ l) in 19 µ l of sterile distilled water or TE in a microcentrifuge tube by heating for 5 min in a boiling water bath; then immediately cooled on ice.
2. Perform the following adddtions on ice:

1 µ l 500 µ M dATP

1 µ l 500 µ M dGTP

1 µ l 500 µ M dTTP

20 µ l 2.5x Random Primers Solution

7 µ l distilled water

1. Mix briefly.
2. Pipette up and down a tip of alpha - 32 P with the above mixed solution until the reddish color on pipette tip disappears.
3. Add 1 µ l Klenow fragment, centrifuge briefly.
4. Incubate at 37 o C for 4 hrs or until it is ready to be added into the prehybridization solution.
5. Denature probe at 90 - 100 o C for 10 minutes then add immediately into the solution in Hybaid tube.
6. Continue with hybridization overnight.

 Autoradiography:

1. After final wash, remove membrane from tube, blot dry with paper towel and wrap with Saran cling film.
2. Monitor radioactivity of the membrane to estimate exposure times.
3. Place the membrane in a film cassette and put X-ray film on both sides of the membrane. This is to be done in the dark room.
4. Store cassette in –20 o C freezer overnight (500 counts/sec) or a week (10 counts/sec) depending on the radioactivity counts.
5. When ready, take out cassette, thaw to room temperature and then develop film.