SOUTHERN TRANSFER - SPONGE METHOD

• I:
  0.25 N HCl

  21.5 ml conc. HCl (11.6 N)
  

  to 1 liter volume.

  II:

  1.5 M NaCl

  87.66 g NaCl (MW 58.44)
  0.5 M NaOH
  20.00 g NaOH (MW 40.00)
  to 1 liter volume.]

  III:

  1.0 M Tris

  121.1 g Tris base (MW 121.1)
  1.5 M NaCl
  87.66 g NaCl (MW 58.44)
  pH to 8.0 with conc. HCl (~50 ml)
  bring to 1 liter volume.

  IV:

  20X SSC

  175.3 g NaCl (MW 58.44)
  88.2 g Na3citrate (MW 294.1)
  pH to 7.0 with conc. HCl (~1 drop)
  bring to 1 liter volume.

1. Take picture of uv irradiated ethidium bromide stained gel. Expose film to uv irradiated gel at f 8.0 with yellow filter (~4 sec. Type 55 Polaroid film, ~2:15 min. Type 57 film, develop negative in 18% Na2SO3 (MW 120.0)).
2. Carefully transfer gel to glass baking dish. Soak gel for the following times in the following solutions:
   15 min. in 0.25 N HCl (Sol'n I).
   15 min. in 0.5 M NaOH, 1.5 M NaCl (Sol'n II), slow agitation.
   15 min. in 0.5 M NaOH, 1.5 M NaCl (Sol'n II), slow agitation.
   30 min. in 1.0 M Tris (pH 8), 1.5 M NaCl (Sol'n III), slow agitation.
   30 min. in 1.0 M Tris (pH 8), 1.5 M NaCl (Sol'n III), slow agitation.
3. Cut 1 Whatman filter paper to size slightly larger than agarose gel.
   Cut 12 S&S BA85 nitrocellulose to size of agarose gel.
   Cut 13 Whatman filter paper to size of agarose gel.
   Cut several paper towels (34 in. stack) to size of agarose gel.
4. Soak filter paper and nitrocellulose in 2X SSC. Soak blotting sponge in baking dish filled with 20X SSC (Sol'n IV).
   Place in order on top of sponge:
   a. larger cut Whatman filter paper
   aa. (optional nitrocellulose filter for sandwich blot)
   b. agarose gel
   c. nitrocellulose filter
   d. 13 Whatman filter papers cut to size of gel
   e. stack of paper towels
   f. flat surface weighted with about 300-500 g.
   
5. Let DNA transfer for 12-18 hrs.
6. Carefully remove nitrocellulose filter(s) from gel. Mark lanes. Treat filter as follows:
   a. Soak nitrocellulose in 6X SSC for 5 min.
   b. Dry Nitrocellulose on dry Whatman filter paper at room temperature.
   c. Dry nitrocellulose in 80 C vacuum oven (20 psi) for 2 hrs.
   d. Store nitrocellulose at room temperature under vacuum until needed.