Southern Blotting: DNA Transfer1. 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
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% (v/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
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