Southern Blotting & Hybridization

This page is just for a personal note for southern blotting and hybridization.

Southern blotting

Depurination: 0.2N HCl for about 10 min (until bromophenol blue turns yellow). To make 100 ml of 0.2N HCl, add 1.66 ml of hydrochloric acid into 99 ml of H2O.

Denaturation: 1.5 M NaCl, 0.5 M NaOH for 45 min.

Neutralization: 1 M Tris-Cl (pH 7.2), 1.5 M NaCl. For 30 min in 1x, then 15 min in 0.6 x.

Transfer buffer: 10x SSC (bottom) and 2x SSC (top).

Hybridization solution

Maleate buffer

100 mM Maleic acid
150 mM NaCl
Adjust pH with NaOH. Autoclave. Store at room temperature.

Blocking stock solution

Dissolve blocking reagent (Roche Applied Science, cat#1096176) in maleate buffer to a final concentration of 10% (w/v) with shaking or heating. Autoclave. Store at -20 C.

(Pre)hybridization solution

1% (w/v) blocking reagent
0.1% (w/v) N-lauroylsarcosine
0.02% (w/v) SDS
(Formamide may also added to 50% (v/v). In this case, increace the final concentration of blocking reagent to 2% (w/v))

To index