Southern Blot Hybridization Protocols
Commonly Used Reagents and Buffer Solutions for Southern Blot
3M ammonium acetate
- Dissolve 231.24g of ammonium
acetate in 1 liter dH2O.
10X TBE
- 100 gm/L (0.89M Tris) MW 121.14
- 55 gm/L (0.89M Boric
acid) MW 61.83
- 40ml of 0.5M EDTA disodium
Adjust pH to 8.0 with 1:10 diluted HCl.
20X SSC
- 3M NaCl
- 0.3M sodium citrate
100X Denhardt's
Solution
- 2% bovine serum albumin
- 2% polyvinylpyrrolidone [Sigma]
- 2% ficoll
Denaturing Solution
DNA Loading Dye
- 15%
ficoll [F-4375 Sigma]
- 10% glycerol
- 0.05% bromophenol blue
- 1X TBE buffer
Weigh 15g of ficoll and add 50-100mg of bromophenol blue,
then add 10ml of glycerol and 90ml of 1X TBE.
Place this mixture on a hot plate with a magnetic stirrer and allow to dissolve and
make up to 100ml with 1X TBE.
DNA Lysis Buffer
- 100mM NaCl
- 10mM Tris-HCl pH 8.0
- 25mM EDTA (disodium salt)
- 0.5% of sodium dodecyl
sulphate
- 0.1mg/ml Proteinase K
Ethidium Bromide
- 10mg of ethidium bromide powder
- 1ml of dH2O
Neutralizing Solution
- 1.5M Tris (pH 8.0)
- 1.0M NaCl
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