Electrophoresis

10x MOPS electrophoresis buffer: 0.2 M MOPS (pH 7.0), 20 mM sodium acetate, 10 mM EDTA (pH 8.0). Store at room temperature avoiding light.

10x Formaldehyde gel-loading buffer: 50% glycerol, 10 mM EDTA (pH 8.0), 0.25% (w/v) bromophenol blue.

1.5% Agarose gel containing 2.2M formaldehyde: To prepare 100 ml, add 1.5 g of agarose to 72 ml of H2O. Microwave. Cool to about 55 C. Add 10 ml of 10x MOPS buffer and 18 ml of formaldehyde.

Prearation of samples: Just before electrophoresis, mix the followings.

RNA (up to 20 ug): 1 ul
10x MOPS electrophoresis buffer: 1 ul
formaldehyde: 2 ul
ethidium bromide (200 ug/ml): 0.5 ul

Heat for 1 hr at 55 C. Chill on ice. Add 1 ul of 10x formaldehyde gel-loading buffer.

Northern blotting

Partial hydrolysis: Rinse the gel with H2O. 0.05N NaOH for 20 min.

Neutralization: 20x SSC for 40 min.

Transfer buffer: 20x 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

5X SSC
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))

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