RT

• CTAB


  • At low ionic strength, it precipitates nucleic acid and acidic polysaccharides
  • At high ionic concentration, it binds to polysaccharides and forms complexes that are removed during chloroform extraction
  • It denatures or inhibits the activity of proteins and/or enzymes
• NaCl


  Helps remove proteins bound to DNA<|>Keeps proteins dissolved in the aqueous layer so they do not precipitate with DNA<|>Provides ionic strength for CTAB to precipitate polysaccharides
• Tris


  An effective buffer between pH 7-9 that increases the permeability of the cell wall and maintains the pH of the solution
• EDTA


  Chelates divalent cations like Mg2+ and Ca2+, reducing the activity of enzymes like DNase and RNase that degrade DNA
• β-Mercaptoethanol


  Reduces disulfide linkages in proteins, denaturing them
• Plant cells have a complex polysaccharide cell wall, with cellulose as a major constituent
• Cell membranes are composed of a diverse set of phospholipid molecules and proteins
• Surfactants like CTAB are amphipathic and can dissolve biological membranes
• Osmosis plays a significant role in cell lysis - hypotonic solutions cause swelling and bursting, while hypertonic solutions lead to plasmolysis
• Divalent cations like Mg2+ and Ca2+ are cofactors for enzymes and are involved in aggregation of nucleic acids with proteins, and cell wall stability
• Disulfide bonds


  Covalent bonds formed between two sulfur atoms in proteins
• β-mercaptoethanol


  A reducing agent that can cleave disulfide bonds in proteins
• Protein denaturation by β-mercaptoethanol

  1. Cleaving disulfide bonds

  2. Disrupting protein structure

  
• Tris buffer


  A buffer solution used to maintain pH in biochemical applications
• Titration of Tris base solution with HCl

  1. Produces Tris weak base

  2. Produces Tris conjugate acid

  3. Results in a buffer near physiological pH range

  
• EDTA


  A chelating agent that binds divalent cations like magnesium and calcium
• EDTA chelation of divalent cations

  1. Binds free divalent cations

  2. Makes them unavailable for enzyme activity and cell processes
• β-Mercaptoethanol


  A strong reducing agent used to remove phenolic compounds from plant extracts
• Protein denaturation by β-mercaptoethanol

  Reduces disulfide bonds<|>Makes proteins insoluble
• PVP


  A polymer used to remove polyphenolic contaminants from plant DNA extracts
• Inhibition of DNase enzyme

  1. Incubating cell lysate in CTAB buffer at 65°C

  2. Allows cooling to room temperature before further processing
• Phenol


  An organic solvent used to remove proteins and polysaccharides from DNA extracts
• Phenol-chloroform extraction


  1. Partitions nonpolar components into phenol phase

  2. Leaves polar DNA in aqueous phase

  3. Denatures proteins
• Chloroform


  A nonpolar solvent that promotes partitioning of lipids and cellular debris into the organic phase
• Isoamyl alcohol


  Added with chloroform to prevent emulsification and foaming during extraction
• RNase A


  An enzyme that hydrolyzes RNA, removing RNA contamination from DNA extracts
• RNase A catalytic mechanism

  1. Cleaves phosphodiester bonds in RNA

  2. Relies on histidine and lysine residues in active site

  3. DNA is not cleaved due to lack of 2'OH group

  
• Isopropanol/ethanol


  Alcohols used to precipitate DNA out of the extraction solution
• DNA precipitation by alcohol

  1. Breaks hydration shell around DNA

  2. Allows DNA to aggregate and precipitate
• Sodium acetate/other salts


  Salts used to neutralize charges on DNA backbone and aid precipitation

  
• Role of salt in DNA precipitation

  1. Dissociates into ions

  2. Sodium ions shield negative charges on DNA

  3. Allows DNA to aggregate and precipitate
• Dielectric constant


  A measure of the ability of a material to store electrical energy in an electric field
• Isopropanol has a lower dielectric constant than ethanol (2–3 volume) and also requires a fair amount of salt to work
• RNA


  Has extra 2'OH that remains hydrogen bounded with water more strongly than DNA, thus tends to stay soluble in it
• Selective precipitation of DNA


  Can be done as DNA is less soluble than RNA
• Isopropanol dissolves nonpolar solvents such as chloroform, thus the impurities from previous step can also be removed
• Using ice-cold isopropanol is generally practiced, but many researchers say that it should be used at room temperature, otherwise it will precipitate polysaccharides also
• Though the yield of DNA will be increased at low temperature, it may increase impurities
• Salts used in DNA extraction

  • Sodium acetate
  • Ammonium acetate
  • Potassium acetate
  • Sodium chloride
  • Lithium chloride
  • Potassium chloride

  
• Role of salt in DNA extraction


  To neutralize the charges on the sugar phosphate backbone of the DNA