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

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

3.31g agarose
230 ml ddH20 (ca. 30 ml will evaporate during heating)
Microwave, 6 minutes, power level 7 (until 30 ml evaporates)
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).
Avoid loading few outer lanes.

Day Run:
Run at 100 V, 30-45 minutes
Run at 200 V, 6 hours
For globin reporters run at 200V for 8 hours until bromphenol blue runs off the gel. At this point xylene cyanol will be 14 cm from bottom. Cut 1 cm off the bottom. Cut 2-3 cm above xylene cyanol for gel piece 11-15 x 20 cm

Overnight Run:
Run at 80 V, 15 hours
Increase to 200 V in the morning until bromphenol blue runs off the gel. At this point xylene cyanol will be 14 cm from bottom.

Place gel on large piece of glass
Cut 1 cm off the bottom. Cut 2-3 cm above xylene cyanol for gel piece 11-15 x 20 cm
Cut small piece off bottom left of gel for orientation.
Place small piece of glass on top of gel and flip gel

Wash gel 30-60 minutes in 10 x SSC, slowly agitating.

Transfer #1 (best for 15 x 20 cm gels)
Set up capillary transfer using 10 x SSC.
Place a glass plate over the tray with 10 x SSC.
Place a piece of 21 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 21 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. Add few ml of 10 x SSC.
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) adding a few ml of 10 x SSC between each layer.
Place 4 pieces of parafilm, one on each side of the gel, to block buffer from transferring "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 x SSC) 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 correctly). Mark membrane with pencil for orientation.
Place the 12 x 20 cm gel (keep it wet!) on top of the membrane (bottom 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 (few seconds).
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 membrane 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 in eppie tube and warm in hybridization oven).
Incubate in hybridization oven, ≥1.5 hr at hybridization temperature.
Add 1/5 of 50 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

KT 050413