Northern Blotting (Breeden Lab)

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)