Principles of Dna Isolation & Purification
Principles of Dna Isolation & Purification
Buffer solutions
Chelation
Salting Out
Basic Principle
• Sampling
• Cell suspension
• Cell lysis
• Purification
• Precipitation
List of chemicals used in DNA extraction
• Tris
• EDTA
• SDS
• Proteinase K
• NaCl
• Phenol
• Chloroform
• Isoamyl Alcohol
• Isopropanol
• Ethanol
The Mammalian Blood
• Red Blood Cells. Anucleate. about 4 million to 6
million per cubic millimeter (microliter) of blood.
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EDTA (Ethylenediaminetetraacetic acid)
• Chelating agent
• amine groups
EDTA As Anti-Coagulant
• anticoagulant
• Tetradenate ligand
• Can be problematic
• pH 8.0
Tris trishydroxymethylaminomethane
• Molecular formula;
C4 H11 NO3
• Biological buffer.
• An alkalizer
SDS Sodium dodecyl sulfate
• Chemical formula;
C12H25NaO4S
• “ionic surfactant
• Osmotic dehydration
Proteinase K Hammers away
Proteins
• Tritirachium album
OR
• phenol
• Chloroform.
• Isoamyl alcohol
summary
• Sample for DNA extraction
• Lysis of cells at elevated temperature +
detergent + enzyme in salt buffer
• Removal of cellular proteins
• Precipitation of nucleic acids with ethanol
• Quantitation and purity measurement of
DNA