1. Depurination of DNA fragments: Wash gel in 0.25 M HCl 5' (small gels), 7' (large gels)

2. Denaturation: Wash gel in (0.5 M NaOH, 1.5 M NaCl) for 20' (small) to 30' (large).

3. Neutralization: Wash in (0.5 M Tris pH 7.0, 3.0 M NaCl) for 20' (small) to 30' (large).

4. Cut nylon membrane (MSI 0.45 micron #N04HY00010) and several pieces of blotting (e.g. Schleicher and Schuell GB002) paper to the same size as the gel. Wet the nylon with dH20, then soak in 5x SSC.

5. Assemble sandwich:

a. Large sheet of plastic wrap

b. Two pieces blot paper (precut to same size as gel) soaked in 20x SSC

c. Gel (wells-side down)

d. Presoaked nylon

e. One piece blot paper soaked in 5x SSC

f. 10-15 pieces of dry blot paper

h. Wrap whole sandwich in the plastic wrap

i. Place glass plate and weight on top and let transfer >3 hr.

6. Crosslink after blotting.

OR

1. Set up a standard alkaline Southern transfer onto a nylon membrane with 0.4 M NaOH, 0.6 M NaCl (for positively charged membranes) or 0.25 M NaOH, 1.5 M NaCl (for uncharged membrane). Transfer >3 hours.

2. Crosslink if using an uncharged membrane.

Solutions for Southern Hybridization and Development Hybridization

20x SSC:

3M NaCl

0.3M Na-citrate; pH7.0

Hybridization Sol'n:

5x SSC

0.5% (w/v) Blocking Reagent

0.1% (w/v) N-lauroylsarcosine, Na-salt

0.02% (w/v) SDS

50% (w/v) formamide

*Blocking reagent does not dissolve rapidly. Heat to ~50-70℃; the sol'n remains turbid. Make ~1 hour in advance. Store hybridization solution at 4℃in a bottle or at -20℃in 50 ml conical tubes.

Note: Prehybridization solution is the hybridization solution without the labelled probe.

Immunochemiluminescence:

Blot Wash #1:

2x SSC; 0.1% (w/v) SDS.

Blot Wash #2:

0.1x SSC; 0.1% (w/v) SDS.

Buffer #1:

100mM maleic acid, pH 7.5

150mM NaCl

Buffer #2:

2% Blocking Reagent in Buffer #1

Heat to 50-70℃; prepare ~60' in advance; sol'n remains turbid

Store at 4℃. Add NaAzide for long term storage.

Buffer #3:

100mM Tris-Cl, pH 9.5

100mM NaCl

50mM MgCl2

Antibody:

10 µl anti-digoxigenin-AP Fab fragments (Boehringer-Mann cat#1093274)

40ml of Buffer #1

10ml of Buffer #2

This will last several months of reuse if stored at 4℃.