Yu:Agarose Formaldehyde

Electrophoresis

1. clean gel box with NaOH and/or SDS, 2 hours to overnight, rinse with water
2. prepare Agarose gel solution [1 % Agarose, 1 x MOPS, H2O to 95 % of end volume]
3. microwave until completely dissolved
4. cool down to 60-70 °C, add Formaldehyde (37 %) to 0.6 M end concentration, pour immediately
5. allow gel to harden at least 30 min
6. prepare running buffer: 1x MOPs, 0.2 M Formaldehyde

Sample preparation

1. use 5-10 µg total RNA per lane (up to 30 µg)
2. bring RNA samples to equal volume (5-10 µl) with DNase/RNase free H₂O, add an equal volume of loading buffer
3. add 0.5 µl EtBr (0.5 µg/µl)
4. heat for 5 min @ 90 °C, cool on ice

Gel run

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

Northern transfer of RNA

1. soak gel 3 times 5 min in distilled water (to remove Formaldehyde)
2. photogragh gel with ruler beside it
3. cut GeneScreen membrane (Nylon, DuPont) to exact gel size
4. soak membrane in water for a few seconds
5. set up capillary blot with 10x SSC transfer buffer
   1. 2 wet Whatman - gel - membrane - 2 wet Whatman - 2 dry Whatman – paper towel - glassplate - weight

 Important: Roll out any air bubbles before applying dry Whatman

1. transfer 16-24 h, changing the paper towel if necessary
2. mark lanes, remove membrane, wash briefly in 2x SSC
3. place membrane on wet Whatman paper and UV-crosslink damp (auto crosslink setting, 254 nm, Stratagene, Stratalinker) or bake membrane @ 80 °C for 1-2 h

Hybridization

1. Prehybridize membrane for 1-4 h @ 42 °C with 5-10 ml prehybridization buffer
2. Heat radioactive labeled probe for 3 min @ 95 °C, cool on ice
3. Discard prehybridization buffer, add hybridization buffer and probe, incubate ON @ 42 °C
4. Wash membrane 1 x 15 min with 2 x SSC @ RT
5. Wash with 2 x SSC, 0.1 % SDS @ 65 °C until background is low
6. Wash with 0.1 x SSC, 0.1 x SDS @ 65 °C (optional)
7. Expose wet membrane under saran wrap (-80 °C)

 Important: never let the membrane dry (until the blot is stripped)

Stripping and re-hybridization

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