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

~undefinedBlocking reagent does not dissolve rapidly. Heat to ~50-70° C; the sol'n remains turbid. Make ~1 hour in advance. Store hybridization solution at 4° C in a bottle or at -20° C 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° C; prepare ~60' in advance; sol'n remains turbid
Store at 4° C. 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° C.

CSPD Ready-to-Use:
Keep reused portions separate from used.
Store @ 4° C PROTECT FROM LIGHT!
[This product is cheaper from Tropix, catalog # CD1000R - this is a 1L bottle of the stuff ...that volume is not cheap. It comes in smaller volumes, depending on your usage.]

NOTES:
All solutions should be at room temp when used on blots.

We reuse prehybridization solution, probes, Buffer 2, antibody conjugate, and CSPD Ready-to-Use. As needed, we replace the prehybridization solution, Buffer 2 and CSPD with fresh solutions. The probes and antibody solutions are used for several months and spiked or made fresh as needed. For most probes, only half of the labelling mixture is needed for sufficient signal. Recipes are from the Boehringer Mannheim Nonradioactive DNA Labeling and Detection Kit and protocols.

Miscellaneous Lab Lore:

1. Fragments with incorporated alkali-labile dig nucleotides may be stripped from blots; the alkali-stable versions do not strip off well.
2. The blocking reagent (Buffer 2) seems to work better after a couple of uses. In the first couple of uses, background spots may be abundant.
3. Depurination is apparently unnecessary for the transfer of small (<10 kb) fragments.
4. Crosslinking is unnecessary if the blot is not going to be stripped and re-probed.
5. There is apparently enough blocking reagent in the antibody solution that the prior blocking step can be skipped without increasing background.
6. If the the half-life of your working CSPD solution begins to decrease rapidly in storage, try using a new storage container (e.g., 50-ml Falcon tube); storage bottles reused many times apparently accumulate something that affects CSPD stability.