Southern Blot
Southern Blot
Southern Blot
Applications
Table of Contents
• Southern Blot Definition
• Principle of Southern Blot
• Requirements
o Equipment
o Materials
o Solutions and Buffers
• Procedure of Southern Blot
o a. Restriction digestion of DNA
o b. Electrophoresis
o c. Denaturation
o d. Blotting
o e. Baking/ Immobilization
o f. Hybridization
o g. Detection
• Result Interpretation of Southern Blot
• Applications of Southern Blot
• Limitations of Southern Blot
• References
• Sources
Requirements
Equipment
• Water Bath
• Agarose gel
• Power supply
• UV radiation
• Hybridization Oven
• Hybridization Bottles
• Trays
• Film processor
• Pipettes
• Centrifuge Tubes
• Glass Plate
• Whatman 3 mm chromatography paper
• Nylon membrane/ Nitrocellulose membrane
• Syringe
• Cellulose acetate membrane
Materials
• Restriction Enzymes
• Restriction enzymes buffer
• Agarose
• TBE buffer
• DNA loading buffer
• Tris base
• Sodium chloride
• Sodium hydroxide
• Sodium citrate
• DNA labeling kit
• Nucleic acid detection kit
• Sodium dodecyl sulfate (SDS)
• Polyvinylpyrrolidone
• Bovine serum albumin
• Formamide
• Phenol
Solutions and Buffers
• Denaturation buffer: NaOH and NaCl in the ratio 1:6
• Neutralization buffer: Tris-HCl and NaCl in the ratio 5:3
• SSC: 175.3 g of NaCl and 88.2 g of Sodium citrate to 1L of distilled water.
• Detection Buffer: Tris-HCl and NaCl in the ratio 5:1.
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