Analysis of Genomic DNA by Southern Hybridization

• Probe preparation
• Filter preparation
• Filter suppression (pre-hybridization)
• Probe suppression
• Washing
• Exposure

Probe preparation

1. Select several independent BAC clones containing the same inserts that will be used as a probe.
2. Confirm the BAC clone integrity using by comparing HindIII digestion patterns.
3. Purification of the whole inserts from those clones. (NotI digestion, PFGE running)
4. Heat gel fragment (FMC SeaPlaque GTG Agarose) containing the insert at 65°C for 10 min. Transfer 25µl to a new 1.5ml tube, boil for 5min. and chill on ice.
5. Add the labelling pre-mix to the tube (Amersham Multiprime DNA labelling system RPN16000Z). Keep overnight at room temperature.

               pre-mix:   4.0µl dCTP 	              4.0µl dGTP 	              4.0µl dTTP 		      5.0µl 10x buffer 		      5.0µl primer/BSA 		      2.5µl [32P]dATP 		      2.0µl Klenow 

6. Remove unincorporated dNTP by spin column (Pharmacia Sephadex G-25)

Filter preparation

1. Digest an appropiate amount of BAC DNA and high molecualr weight genomic DNA. For Southern hybridization, approximately 10µg of DNA should be loaded into each slot of the gel when probes of standard length and high specific activity are used to detect single copy sequences.

      Example: Human genomic DNA (Clonetech)    1) Mix DNA 100µl(10µg), NEBuffer2 40µl, HinIII(20u/µl) 2.5µl,       incubate at 37°C for 2 - 2.5 hours   2) Add 800µl absolute EtOH to the tube,       and invert for several times.   3) Vortex with highest speed for 2 min.   4) Spin at the highest speed for not longer than 5 min.   5) Remove the EtOH completely and dissolve with 25µl 2x BPB. 

2. Load both the BAC clone and human genomic DNA onto 0.7% gel in 0.5x TBE. The gel should be run very slowly to prevent smearing of the DNA fragments.
3. After electrophoresis, stain the gel with EtBr and photograph ir. Place a transparent ruler alongside the gel so that the distance of migration of the DNA bands can be read directly from the photograph.
4. Capillary transfer the gel to the positively charged nylon filter (Schleicher & Schull, NYTRAN PLUS, 0.45µm pore size).

       Denaturation and transfer solution: 0.5M NaOH + 1.5M NaCl    Neutralizing solution: 0.5M Tris + 1.5M NaCl (pH7.4) 

5. To fix the DNA on the filter, bake the membrane for 1.5-2 hours at 80°C in the vacuum oven.

Filter supression (pre-hybridization)

1. Place the filter in the Falcon #2098 plastic centrifuge tube.
2. Add 4ml hybridization solution to the tube (1M NaCl. 0.05M Tris·Cl pH8.0, 0.5mM EDTA, 1% SDS, and 10% Dextran).
3. Boil 40µl (10mg/ml) cold nonspecific DNA (Sigma, human placental DNA) in 400µl TE (pH8.0) for 5 min. and add to the hybridization buffer.
4. Hybridize the filter with cold nonspecific DNA at 65µC for overnight in a hybridization incubator (Robbins Scientific).

Probe supression

1. Prepare the supression mixture

       10µl labelled BAC probe    40µl cold nonspecific DNA    50µl poly dG-dT/dA-dC (0.25mg/ml) (Pharmacia)   200µl 1.5x SET (1x SET: 0.6M NaCl, 0,02M EDTA,          0.2M Tris·Cl [pH8.0], 2% SDS,         0.1% sodium pyrophosphate) 

2. Boil for 5 min.
3. Keep at 65°C for at least 3.5 hours.
4. Add this mixture to the pre-hybridized filter, keep at 65°C overnight.

Washing

Wash the filter several times at 65°C with sufficient volumes of 0.1x SSC/0.1% SDS (20 min. each time for at least twice).

Exposure

Expose the filter with phosphoimgaer or X-ray film (Kodak BIOMAX MS film and intensifying screen) until signal is visible.