DNA Southern Blot Protocol

Southern Blot Hybridization Protocols

Protocol 2. Protocol for Southern Blotting of Genimic DNA

Protocol 3. DNA Analysis by Southern Blotting

Protocol 4. Southern Blot Analysis

Protocol 4. Southern Blot Method



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
0.5M NaOH
1.0M NaCl
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