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Southern Blotting
| Note: We usually use Immobilon N membranes which
can be used 15-20 times. Other membranes (like nylon membranes)
can be used as well, but may not give the same number of re-uses. |
1. Cut off the upper left corner of the gel. Flip the gel
so comb openings face down.
2. Denature gel for 30 min. in 1X Denaturing solution with gentle shaking;
treat each gel in about three times its volume of solution – 1 L/gel.
3. Neutralize gel for 30 min. in 1X Neutralizing solution with gentle
shaking; treat each gel in about three times its volume of solution –
1 L/gel.
4. Cut membranes to the same size as the gel (20 x 23 cm). Snip upper
left corner of membrane. Label membrane with a permanent, alcohol-resistant
marker.
5. Prepare transfer trays: rinse sponges in dH2O and squeeze gently. Place
two matching sponges in the tray containing2.5 1 transfer buffer (5x SSC).
Soak sponges thoroughly in transfer buffer.
6. Wet Immobilon membranes in 95% EtOH, rinse in ddH2O ~10 min., and store
in 5X SSC until ready for use.
7. Dip one sheet of gel blot paper in transfer buffer and place on sponges.
8. Place gel onto blotting paper; the cut-off corner has to be at the
upper right.
9. Place the membrane on gel, cut-off corner at the upper right.
10. Place one sheet of pre-wetted gel blot paper on membrane.
| Note: Make sure that there are no air bubbles
between gel blot paper, gel, and membrane. Roll out any bubbles
between each layer with a glass pipette or a plastic tube. |
11. Place plastic sheets around the edges of the gel blot
paper to avoid paper towels wicking transfer buffer form anywhere other
than directly through the gel. 12. Place stack of paper towels on top
of transfer system; place gel tray on top of paper towels, and place two
half-full 500 ml flasks (or another weight) on the gel tray.
13. Transfer 12-18 hours.
14. Remove membrane and rinse for 10 min. in 2X SSC with shaking.
15. Air dry membranes; bake between filter paper at 80 C with vacuum set
at 15 for 2 hours. Store membranes between clean filter paper in Ziploc
bags.
| Note: You can check the transfer under UV light;
DNA will appear pink-orange. |
1X Denaturing
Solution – 0.2 N NaOH, 0.6 M NaCl
8.0 g NaOH
35.0 g NaCl
ddH2O to 1 liter
1X Neutralizing Solution – 0.5 M Tris-7.5,
1.5 M NaCl
63.5 g Tris-HCl (MW=156.60)
11.8 g Tris-base (MW=121.10)
87.6 g NaCl
ddH2O to 1 liter
Loading Dye
15% Ficoll type 400
0.25% Bromophenol Blue
0.25% Xylene cyanol (optional)
dissolve in ddH2O
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