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Southern Blot 1. Run gel (0.8 -1.0% agarose is best). For yeast chromosomal Southerns, digest 20 g DNA. Use 50
 

Summary: 41
Southern Blot
1. Run gel (0.8 -1.0% agarose is best). For yeast chromosomal Southerns, digest 20 g DNA. Use 50
ml minigel for most purposes. Photograph gel, but minimize exposure to UV light.
2. Depurination: Place gel in 250 mls 0.25M HCl.
(250 mls = 11 mls 5.8M HCl + 250 mls ddH2O)
Agitate gently on rotator for 10 minutes after dye front turns yellow.
3. Decant HCl. Wash briefly with ddH2O.
4. Denaturation: Place gel in 250 mls 1.5M NaCl + 0.5M NaOH.
(500 mls NaCl/NaOH = 44 g NaCl + 10 g NaOH)
Rotate gently 20 minutes.
Decant, add 250 mls fresh NaCl/NaOH, rotate 20 minutes.
5. Decant NaCl/NaOH. Wash briefly with ddH2O.
6. Neutralization: Place gel in 250 mls of 3M NaCl + 0.5M Tris, pH 7.0.
500 mls = 88 g NaCl + 3 g Tris base + 36 g Tris HCl
Rotate gently 20 minutes.
Decant, add 250 mls fresh NaCl/Tris, rotate 20 minutes.
7. Measure gel exactly. Cut off bottom right corner. Put gel back in neutralization solution. Cut
nitrocellulose paper about 1 mm shorter than gel and wet in ddH2O. Soak nitrocellulose paper 5-
10 minutes in 20X SSC. Cut 2 sheets of THIN filter paper 1 mm shorter than nitrocellulose paper.

  

Source: Aris, John P. - Department of Anatomy and Cell Biology, University of Florida

 

Collections: Biology and Medicine