Northern Blot

Northern Blot

 

  I. Electrophoresis

• clean gel box with NaOH and/or SDS, 2 hours to overnight, rinse with water
• prepare Agarose gel solution [1 % Agarose, 1 x MOPS, H 2 O to 95 % of endvolume ]
• microwave until completely dissolved
• cool down to 60-70 °C , add Formaldehyde (37 %) to 0.6 M endconcentration, pour immediately
• allow gel to harden at least 30 min
• prepare running buffer [1 x MOPS, 0.2 M Formaldehyde]

 II. Sample preparation

• use 5-10 µg total RNA per lane (up to 30 µg)
• bring RNA with H 2 O DEPC to equal volume (5-10 µl), add same vol. loading buffer
• add 0.5 µl EtBr (0.5 µg/µl)
• heat for 5 min @ 90 °C , cool on ice

 III. Gel run

• run gel (8 x 10 cm) in fume hood with 70-100 V (-> 50-70 mA)
• run until BPB is near the gel end (2.5-3.5 h)

 IV. Northern transfer of RNA

• soak gel 3 times 5 min in distilled water (to remove Formaldehyde)
• photogragh gel with ruler beside it
• cut GeneScreen membrane (Nylon, DuPont) to exact gel size
• soak membrane in water for a few seconds
• set up capillary blot with 10 x SSC transfer buffer:
  2 wet Whatman - gel - membrane - 2 wet Whatman - 2 dry Whatman - papertowel - glasplate - weight
• transfer 16-24 h with changes of the papertowel
• mark lanes, remove membrane, wash briefly in 2 x SSC
• place membrane on wet Whatman paper and UV-crosslink damp (auto crosslink setting, 254 nm, Stratagene, Stratalinker)
• bake membrane @ 80 °C for 1-2 h

 V. Hybridization

• prehybridize membrane for 1-4 h @ 42 °C with 5-10 ml prehybridization buffer
• heat radioactive labeled probe for 3 min @ 95 °C , cool on ice
• discard prehybridization buffer, add hybridization buffer and probe, incubate ON @ 42 °C
• wash membrane 1 x 15 min with 2 x SSC @ RT
• wash with 2 x SSC, 0.1 % SDS @ 65 °C until background is low
• wash with 0.1 x SSC, 0.1 x SDS @ 65 °C (optional)
• expose wet membrane under saran wrap (-80 °C)
• important: never let the membrane dry (until the blot is stripped)

 VI. Stripping and re-hybridization

• wash membrane for 30 min to 3 h in strip solution @ 75 - 85 °C until no radioactivity can be detected on the membrane
• membrane can now be air dried and stored @ RT
• for re-hybridization (up to 10 times) follow the hybridization protocol

 

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Buffers:

 10 x MOPS:
0.4 M Morpholinopropanesulfonic acid (free acid); 0.1 M Na-acetate-3 x H 2 O; 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 2 O]

 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 2 O]
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]