Southern blot

  1.  Photograph gel.
2.  Treat gel with 2 volumes of 0.2 N HCl and shake 15 min to depurinate DNA.  Pour off diluted HCl.
3.  Treat gel with 2 volumes of 0.4 N NaOH and shake 15 min.
4.  Using gloves, cut two sheets of Whatman filter paper larger than the gel. Cut one sheet of HYBOND-N nylon membrane and two sheets of Whatman filter paper to fit the width of lanes loaded with DNA.
5.  Construct sandwich:
a. sponges soaked in 0.4 N NaOH.
b. 2 X filter paper soaked beforehand in 0.4N NaOH. Carefully roll out bubbles.
c. gel
d. membrane (soaked in 0.4N NaOH). Cut off excess gel. Roll out bubbles.
e. 2 X filter paper (soaked in 0.4N NaOH). Carefully roll out bubbles.
f.  saran wrap. Cover all edges of filter paper and sponge to prevent wicking.
g. stack of paper towels. No contact between towel and sponge.
h. weight.  A small book should work fine.
6.  Let DNA transfer 3-4 hours.  Remove wet paper towels occasionally and add dry towels as necessary.
7.  After DNA transfer, neutralize membrane with 50 mL 2x SSC, 0.2M Tris PH 7.4.
8.  Blot membrane dry and place in glass cylinder preheated to 65oC.  Add FBI hybridization buffer (1.5X SSPE, 10% PEG, 7% SDS) containing 10 μg/ml denatured Herring Sperm DNA.
9.  Incubate at least 15 min (1 hour is recommended) at 65°C.
10.  Denature probe(s) at 100°C for 5 min.
11.  Add appropriate amount of probe to glass tubes (over 106 cpm/ml).
12.  Hybridize at least 2 hours (usually overnight).

13. At end of hybridization, pour off liquid into a radioactive waste container.

14.  Wash four times in 1X SSC; 0.1% SDS. For more stringent conditions, wash at 0.1X SSC; 0.1% SDS (beware that this will remove some signal).

15.  Expose to film.