• 10 X MOPS
  0.2 M MOPS, 10 mM EDTA (disodium), 50 mM sodium acetate, pH=7.0, filter
  sterilize, do not autoclave
  
• 20X SSC
  175.3 gm sodium cloride, 88.2 gm sodium citrate (dihydrate), pH=7.0, q.s. to 1 L.
  
• 37% formaldehyde, pH >4.0
  
• Deionized formamide, kept frozen at -20°C until use
  
• Ethidium bromide (10 mg/ml stock)
  
• 1 M sodium phosphate buffer, pH=7.0
  
• 10% SDS
  
• 50X Denhardts
  10 gm Ficoll, 5 gm BSA (bovine serum albumin), 5 gm PVP (polyvinylpyrrolidone),
  50 mg NaN3. q.s. to 1 L
  
• 50% Dextran sulfate
  
• 1X Blot Wash
  
• 1X SSC, 0.1% SDS, 0.1% sodium pyrophospate.

RNA gel

• Pour 1% denaturing RNA gel (examples shown below)
  
  1. Dissolve agarose in DEPC-treated water by boiling, cool to 56°C.
     
  2. Combine 10X MOPS and 37% formaldehyde (pH greater than 4.0) together and warm to 56°C.
     
  3. Gently swirl the melted agarose together with the MOPS/formaldehyde, and immediately pour gel, using the smallest and thinnest comb possible for the amount of RNA.
  4. Once solidified, immerse gel in 1X MOPS running buffer while RNA is prepared to load.
     
     Quantities for pouring gel:

  Total gel volume (ml):	150	200	250
  Agarose (gm):	1.5	2.0	2.5
  37% formaldehyde (ml):	26.8	35.7	44.6
  10X MOPS (ml):	15.0	20.0	25.0

• Prepare 5-40 µg total RNA (from stock stored in EtOH)
  
  1. EtOH precipitate desired amount of RNA (our standard amount is 10 µg, loaded in 23 µl).
     
  2. Wash once with 70% EtOH (made with DEPC-treated water).
  3. Dry pellet briefly at room temperature for 10 minutes. (Do not overdry!!)
     
  4. Resuspend pellet in 4.5 µl DEPC-treated water.
     
  5. Heat sample to 65°C for 5 minutes.
     
  6. Add loading buffer: (per sample)

     2.0	µl	10X	MOPS
     3.5	µl	37%	formaldelhyde
     10.0	µl	d.i.	formamide
     3.0	µl	EtBr	(1 mg/ml)

  7. Heat sample to 65°C for 5 minutes.
     
  8. Add tracking dye and load gel immediately, or hold samples on ice, place standards on outside lanes.
     
  9. Run gel overnight at 20 V, or during the day at 60 V until the dye front migrates half-way down the gel.

1. Cut off lanes containing standards and stain in EtBr for 15-30 minutes.
   
2. Destain standards in d.i. water for at least one hour.
   
3. Examine the quality of RNA by visualizing with uv, and mark the standards with India ink on the gel to be transferred. We also mark the location of the ribosomal bands.
   
4. Rinse the gel thoroughly with d.i. water to remove excess formaldehyde.
5. Soak the gel in 20X SSC while preparing the transfer apparatus (using 20X SSC and Whatman wick).
   
6. Blot onto either 0.45 µm nitrocellulose or nylon membranes (nylon can be stripped and reprobed with less wear than nitrocellulose).
   
7. Pre-wet the membrane with d.i. water and soak in 20X SSC for 5 minutes.
   
8. Apply membrane to gel on transfer apparatus (roll out the air pockets), cover with three pieces of 3M Whatman (wet briefly with 20X SSC) and paper towels, and transfer overnight.
9. Prior to hybridization, bake nitrocellulose membrane for 1-2 hours in 80°C vacuum oven.

          Prehybridization solution    Hybridization solution 

H2O                 10.0 ml                   0.0  ml
d.i. formamide      50.0 ml                  37.5  ml
20X SSC             25.0 ml                  18.75 ml
50X Denhardts       10.0 ml                   1.5  ml
1 M NaPO4 pH 7.0     5.0 ml                   1.5  ml
s.s. DNA (10 mg/ml)  2.5 ml                   0.75 ml
10% SDS              1.0 ml                   0.75 ml
50% Dextran sulfate  0.0 ml                  15.0  ml
                   ________                  ________
                   100.0 ml                  75.0  ml 
        

          Prehybridization solution    Hybridization solution 

H2O                 10.0 ml                   0.0  ml
d.i. formamide      50.0 ml                  37.5  ml
20X SSC             25.0 ml                  18.75 ml
50X Denhardts       10.0 ml                   1.5  ml
1 M NaPO4 pH 7.0     5.0 ml                   1.5  ml
s.s. DNA (10 mg/ml)  2.5 ml                   0.75 ml
10% SDS              1.0 ml                   0.75 ml
50% Dextran sulfate  0.0 ml                  15.0  ml
                   ________                  ________
                   100.0 ml                  75.0  ml 

1. Pre-hybridize blot at 42°C for at least 6 hours, but preferably overnight, volume used depends on size of the blot.
2. Label probe by standard protocols, with specific activity of at least 1 X 10¹⁰ cpm/ug.
3. Exchange pre-hybridization solution for hybridization solution, boil, and add probe.
4. Hybridize overnight at 42°C.

Washing stringency depends on probe, and should be increased or decreased accordingly. Standard conditions follow:

1. Rinse blot in 1X blot wash (1X SSC, 0.1% SDS, 0.1% sodium pyrophosphate) for 5 minutes at room temperature.
2. Wash three times in 1X blot wash at 68°C for 20 minutes each, and monitor radioactivity, if background remains high, wash in 0.1X blot wash (0.1X SSC, 0.1% SDS, 0.1% sodium pyrophosphate) for 10 minutes, monitor closely during this wash.
3. Expose blot on film overnight at -70°C, or 2-5 days if blot does not appear to have any detectable radioactivity.