Northern Blotting (Breeden Lab)
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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)
