Northern Blot

Gel:

1.2% agarose/formaldehyde gel (change percentage according to use):

Prepare 2 liters of 1xMOPS, and place in cold room

3.31 g agarose

230 ml ddH20 (ca. 30 ml will evaporate during heating)

Microwave, 6 mins, power level 7.

Swirl

After cooling down a bit, add 27.6 ml 10xMOPS

Add 48 ml Formaldehyde in the hood

Swirl, and pour gel.

Mix RNA samples (only the amount needed for one run!) in Formamide LB 1:1 with 2 x MOPS/formaldehyde LB.

Add 50 ml formaldehyde to the 2 liters of cold 1xMOPS buffer and add to chamber with agarose gel.

Heat samples at 80 °C for 2-5 mins.

Load on agarose/formaldehyde gel (rinse wells before loading).

Run at 100 V, 30-45 mins

When using globin reporters, run at 150 V until bromphenol blue dye is approximately 1 inch from the buttom of the gel (approximately 6 hrs). Cut out piece of gel (for globins between xylene cyanol and bromphenol blue = 11-15 x 20 cm). Wash gel 30-60 mins in 10 x SSC, slowly agitating.

Transfer #1 (best for 15 x 20 cm gels)

Set up capillary transfer using same 10 x SSC used for washing gel.

Place a glass plate over the tray with 10 x SSC.

Place a piece of 20 x 46 cm 3MM paper over glass plate, so it dips into 10 x SSC buffer. Wet paper with 10 x SSC (no puddles, bubbles or wrinkles). Roll over with 10 ml pipette. Repeat with one more piece of 20 x 46 cm 3MM.

Cut gel to size of membrane and place upside down on top of 3MM papers (no bubbles). Mark membrane with pencil for orientation.

Wet a 15 x 20 cm membrane and place on top of gel (no bubbles!). Roll over with a pipette.

Wet a piece of 15 x 20 cm 3MM paper. Drip off and place on top of membrane. Roll over with pipette (no bubbles or wrinkles). Repeat with two more wet 15 x 20 cm 3MM papers (three total).

Place 4 pieces of parafilm, one on each side of the gel, to block buffer from transfering "outside" of gel.

Add three pieces of dry 15 x 20 cm 3MM papers on top of stack.

Add about 10 cm worth of paper towel on top of stack.

Cover with Saran wrap and place a couple of nice lifescience catalogs (not too heavy) on top.

Leave over night for capillary transfer.

Transfer #2 (easier for 12 x 20 or smaller gels)

Set up capillary transfer using same 10 x SSC used for washing gel.

Place a tray with 500 ml 10 x SSC on a suitable support (e.g. two P1000 tips racks) besides a stack of paper towels (nice and even stack). The stack should be as tall as the level of 10 x SSC or higher (i.e. 7-8 cm).

Place 2 pieces of dry 12 x 20 cm 3MM paper on top of the stack of paper towels.

Place 2 pieces of wet (10 x SSC) 12 x 20 cm 3MM paper on top of the stack of paper towels (no bubbles or wrinkles). Roll over with 10 ml pipette. Repeat with one more piece of 12 x 20 cm 3MM.

Place wet (10 xSSC)12 x 20 cm membrane (nitrocellulose or nylon membrane) on top of the stack avoiding bubbles (keep it wet for optimal results and be careful to align corretly). Mark membrane with pencil for orientation.

Place the 12 x 20 cm gel (keep it wet!) on top of the membrane (buttom down). Roll over with 10 ml pipette to remove any bubbles.

Place 2-3 pieces of wet 12 x 20 cm 3MM on top.

Place 2 pieces of wet 50 x 20 cm 3MM as a bridge connecting the stack with the tray containing 500 ml 10 x SSC. Roll over with 10 ml pipette.

Place a glass plate on top of the stack and a 500 ml bottle (containing 300-400 ml) on top of the glass plate to ensure flow.

Leave over night for capillary transfer.

Hybridization:

Wash membrane gently and briefly with water to remove salt.

Place membrane on top of 3MM paper, RNA side facing up, and crosslink RNA to membrane in Stratalinker, using "auto crosslink" setting.

(Membrane can be stored at 4°C in a sealed plastic bag)

Place membrane in hybridization bottle (roll moist membrane around a 25 ml pipette and insert into bottle). If the mebrane is longer than 11 cm use “mesh” to sandwich around the membrane.

Add 20 ml prewarmed hybridization buffer (keep 0.8 ml hybridization buffer for probe).

Incubate in hybridization oven, ≥1.5 hr at hybridization temperature.

Add 1/4 of 10 ml transcription reaction (see in vitro transcription protocol) of RNA probe to 0.8 ml hybridization buffer.

95°C, 2 min.

Add probe in hybridization buffer to hybridization bottle.

Incubate over night at hybridization temperature (60°C for globins).

Wash:

Wash membrane briefly with approximately 100 ml 0.1xSSC/0.1% SDS while in hybridization bottle.

Wash blot 3 x 15 mins at RT with 0.1 x SSC/0.1% SDS (250 ml).

Wash blot 1 x 15 mins at 60°C with 0.1 x SSC/0.1% SDS (250 ml prewarmed in microwave). Check signal with monitor.

Seal membrane in plastic bag or wrap in Saran wrap and expose to film or phosphorimager.

Buffers:

10xMOPS:

0.2M MOPS

40 mM NaAc

5 mM EDTA

Adjust pH to 7.0

Formamide LB:

Deionized formamide

5 mM EDTA

0.1% Bromophenol blue

0.1% Xylene cyanol

2xMOPS/formaldehyde LB (prepare fresh every time):

2xMOPS

30% Formaldehyde

5 mM EDTA

0.1% Bromophenol blue

0.1% Xylene cyanol

Hybridization buffer:

20 ml total

10 ml deionized formamide

4 ml 10 x SSC

1 ml 10% SDS

0.5 ml 1M Na-phosphate pH6.5

0.3 ml 10 mg/ml carrier RNA (denatured at 95°C, 2 min)

2 ml 50 x Denhardts

2.2 ml ddH2O