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Breeden Lab, Northern Blot

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Northern Blot


 I. Electrophoresis

 II. Sample preparation

 III. Gel run

 IV. Northern transfer of RNA

 V. Hybridization

 VI. Stripping and re-hybridization



Buffers:

 10 x MOPS:
0.4 M Morpholinopropanesulfonic acid (free acid); 0.1 M Na-acetate-3 x H
2O; 10 mM EDTA; adjust to pH 7.2 with NaOH; store dark in fridge:
[500 ml: 41.9 g MOPS, 6.8 g NaAc, 10 ml 0.5 M EDTA]

 Loading Buffer:
1 x MOPS; 18.5 % Formaldehyde; 50 % Formamide; 4 % Ficoll400; Bromophenolblue; store at -20 °C:
[1 ml: 100 µl 10 x MOPS, 500 µl Formamide, 185 µl Formaldehyde, 40 mg Ficoll400, Bromophenolblue, 215 µl H
2O]

 Prehybridization-buffer:
5 x SSC; 50 % Formamide; 5 x Denhardt's-solution; 1 % SDS; 100 µg/ml heat-denatured sheared non- homologous DNA (Salmon sperm DNA or yeast tRNA)
[100 ml: 25 ml 20 x SSC, 50 ml Formamide, 5 ml 100 x Denhardt's, 1 g SDS, 1 ml 10 mg/ml DNA]

 Hybridization-buffer:
Prehybridization buffer with 5 % Dextransulfate (Na-salt, MW 500,000, 50 % stock-solution) and without non-homologous DNA

 100 x Denhardt's solution:
[for 500 ml: 10 g Ficoll 400; 10 g polyvinylpyrrolidone MW 360000; 10 g BSA fraction V; H
2O]
store at -20 °C.

 20 x SSC:
3 M NaCl; 0.3 M Na-citrate
[1 l: 175.3 g NaCl, 88.2 g NaCitrate]

 Strip-solution:
5 mM Tris pH 8; 0.2 mM EDTA; 0.05 % Na-pyrophosphate; 0.1 x Denhardt's solution
[500 ml: 2.5 ml 1 M Tris, 200 µl 0.5 M EDTA, 5 ml 5 % NaPP, 1 ml 50 x Denhardt's]