ALTERNATE SOUTHERN BLOT HYBRIDIZATION PROTOCOL

1. Prepare DNA from lymphocytes (peripheral blood, synovial fluid, or synovial membrane) according to the G.T.Williams protocol with cell lysis and restriction endonuclease digestion in agarose.
   
2. Electrophoretically separate restriction enzyme digested DNA in agarose and TBE buffer. UV irradiate the gel for 6 minutes (photograph the gel at the same time) prior to base treatment for 45 minutes and neutralizing for 90 minutes.
   
3. Transfer the DNA overnight to Nytran nylon membranes by the usual Southern blotting technique. After transfer, soak the membranes for 30 minutes in 5x SSPE at room temperature. Remove excess solution with a Whatman 3MM filter paper. Wrap the membrane in Saran wrap and treat with ultraviolet light for 4 minutes. Store at 4°C until needed.
   

1. Discard the prehybridization solution.
   
2. Add 15 ml of fresh hybridization solution and reseal the container. Keep the container at 42°C until the probe is added.
   
3. Add 1.5 mg (150 µl of a 10 mg/ml stock) of sheared, salmon sperm DNA to the labeled DNA probe. Heat the labeled probe DNA and salmon sperm DNA mixture to 95°C for 10 minutes and transfer this denatured DNA to the hybridization container. When using Seal-a-Meal bags, use a 1 ml syringe and a 25 ga needle, and reseal the bag around the injection site.
   
4. Hybridize overnight at 42°C with constant agitation.
   
5. Wash the membranes twice for 30 minutes in wash solution I, and once for 30 minutes in wash solution II.

Base Treatment Solution (1.5M NaCl, 0.5M NaOH) for Southern and Benton Davis blots.

For 2 liters:

175.35 g NaCl

40 g NaOH

QS to 2 liters with water and autoclave.

Neutralizing Solution (0.5 M Tris-HCl, 3M NaCl, pH 7.4) for Southern and Benton Davis blots.

For 2 Liters:

121.1 g Tris

350.7 g NaCl

Adjus t pH to 7.4 with concentrated HCl (c. 70 ml?) and QS to 2 liters with water.

20X SSC (3 M NaCl, 0.3 M sodium citrate, pH 7.0)

175.4 g NaCl

88.2 g sodium citrate dihydrate

Adjust pH to 7.0 with NaOH. QS to 1 L with water. Autoclave.

Hybridization Solution (Gimble lab)

5% dextran sulfate (60 g dissolved in about 200 ml water)

40% deionized formamide (240 ml)

4X SSC (120 ml of 20x SSC stock)

6.7 mM Tris-HCl, pH 7.6 (2 ml of 2M Tris-HCl, pH 7.6 stock)

0.83x Denhardt's solution (5 ml 100x Denhardt's)

2% SDS (5 g)

Add heat denatured salmon sperm DNA to the aliquot to be hybridized to a final concentration of 100 µg/ml

Wash Solution I

Wash twice for 30 minutes at 60°C

300 mM NaCl

60 mM Tris-HCl, pH 8.0

2 mM EDTA

1% SDS

Wash Solution II

Wash once for 30 minutes at 60°C

0.2x SSPE

0.1% SDS