Chapter 4: DNA Isolation: Plasmids, Genomic DNA & CDNA

Extraction and Purification of Nucleic Acids

  • Collect tissue containing nucleic acid of interest
  • Use fresh material, or frozen quickly to maintain quality and limit degradation by nucleases which release during homogenization
  • Homogenize tissue and lyse cells
  • EDTA: Chelates Mg and inactivates nuclease activity since Mg is cofactor for nucleases
  • SDS: Solubilizes membrane and lyse cells
  • Bacterial: Sonification, freeze thaw cycles, enzymes, high pressure
  • Animal/Plant: Homogenizers, Yeast bead breakers
  • Isolate desired component
  • Removal of; RNA: RNase hard to inactivate, DNA: DNase inactivated by heat
  • Protein: Proteinase K, non-specific protease not inactivated by SDS/EDTA add to buffer
  • Phenol extraction: Add equal amounts phenol and mix, phenol is denser than nucleic acid solution and protein solution, non-polar flip outside and protein denatures and can be collected by centrifuge, inactivates RNase and alkaline phosphatase but proteins cannot be recovered
  • Concentration of nucleic acids
  • 0.1 * 3M sodium acetate and 2.5*ethanol and chill, centrifuge and wash with 70% ethanol to remove sale in DNA pellet, can resuspend

Plasmid Purification

Pipette carefully to keep plasmid DNA supercoiled

Cesium Chloride Gradient

High salt solution creates density gradient when ultracentrifuge

  • DNA molecules will migrate to point where they have same density as gradient and form bands (RNA at bottom, Protein up)
  • Ethidium Bromide: Plasmid DNA binds less making them denser, more gDNA than plasmid

Alkaline Denaturation

  • Linear, chromosomal DNA denatured at pH 12, plasmid can renature because interlocked
  • Column Purification: Plasmid DNA binds to beads with positive charge and rest washed off

Quick and Dirty: Heat and remove glob of gDNA with toothpick then ethanol ppt of plasmid DNA in supernatant

Detection and Quantitation of Nucleic Acids

  • Spectrophotometric Quantitation: Purine/pyrimidine bases absorb light at 260 nm (protein-280, alc-230)
  • Concentration of nucleic acid is proportional to absorbance, contaminants alter measurement
  • 1 0D260 = 50 ug/mL (DNA) = 40 ug/mL (RNA)
  • Limitations: Volume needed, expensive nanodrop, contamination (tRNA gDNA), cant evaluate integrity

Ethidium Bromide

  • Dye used to evaluate integrity of ds nucleic acids
  • Stacks between pairs and absorbs UV light and emits this in the form or red-orange light
  • Use gloves or will bind user’s DNA

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