RNA Sample preparation
- Add RNA samples (10µg in nuclease-free water/buffer) to eppi tubes on ice. The more concentrated the RNA is, the better
- Add 1x volume of Ambion Glyoxal sample loading dye (Cat#8551)or MOPs Loading Buffer or Formamide Loading Buffer.
- Treat the RNA markers (Invitrogen, cat#15620-016, 0.24-9.5kb, use 3µl per lane) the same way).
- Incubate samples for 30 mins at 50°C (make sure you use cap-lock for the eppis to avoid any liquid getting into your samples during incubation in the waterbath)
- Cool on ice for 5-10 mins.
- Vortex, pulse-centrifuge to bring the samples down.
- Run the gel - 70 volts for 6 hrs. Dye migrates 1 cm/hr at 50 volts
- Photograph the gel under UV – with a ruler
- Trim the gel down to the essential size and wash 2x in copious amounts of water (in a large plastic tray). Soak in 10X SSC buffer for 15-30 mins.
- Cut long blotter paper to the right length, soak in buffer and fold over a glass plate for a buffer bridge in a glass tray. Place gel on top of it and roll out the bubbles. Place trimmed films around the gel to ensure that vertical capillary transfer only occurs via the gel area.
- Cut Hybond N+ membrane to the exact size/slightly smaller. Wet the membrane in 10X SSC, cover the top of the gel with the membrane and roll out the bubbles.
- Overlay with 2 layers of wet Whatman paper (one layer at a time), roll out the bubbles each time.
- Overlay with 2-2.5” of paper towels (cut to size) or Whatman paper (cut to size). Cover with a glass plate and 1/2 filled 500 ml bottle and level it.
- Transfer with ample 10X SSC in the glass tray overnight.
- Mark the wells with pencil before removing and number the lanes.
- Rinse in 2X SSC, dry on filter paper.
- UV-Crosslink in the Stratalinker – autocrosslink, 1-2x.
- Store blot in SaranWrap or go directly to pre-hyb.
Hybridization and Wash
- Prehyb 15-30 mins at 65°C
- Pre-warm the buffer
- Pre-hyb using the Hyb Buffer
- Boil the probe if dsDNA fragments – on the heat block for 2 mins.
Use CAPS LOCK and caution! Make sure you monitor the area for any radioactive contamination.
- Discard the pre-hyb buffer and add 10 ml of new hyb buffer, add the probe into the hyb tube and hyb O/N at 65°C rotating
- Discard probe (or store in 50ml conical)
- Add 50 ml of 2X SSC/0.1% SDS for a quick rinse. Discard.
- Add approx. 50 ml of 2X SSC/0.1% SDS, 15 mins at 65°C rotating.
- Add approx. 50 ml of 1X SSC/0.1% SDS, 15 mins at 65°C rotating.
- Add approx. 50 ml of 0.5X SSC/0.1% SDS, 15 mins at 65°C rotating
- Wrap in seran wrap (moist but not wet). Expose on the phosphorimager screen
Stripping the Membrane
Boil 500 ml of water (with 2 ml of 20% SDS), add into a tray, put the membrane and gentle rock for 30 mins. Discard water (down the sink is ok), and repeat (2-3x) until small/none of the signal can be detected by Geiger counter.