Lecture 13 - DNA Purification, Gel Electrophoresis, and DNA Visualization

Genome - Complete set of nucleic acids containing genes for organism

Purification of genomic DNA - Starting point for gene cloning, gene libraries, PCR, hybridizations, RFLP, VNTR, or sequencings

DNA purified using organic solvents

Reagents used to extract/purify DNA

• NaCl

• Ethanol

• Phenol

• Chloroform

• Centrifugation

DNA purification techniques

• Crude Lysate —> Heating, add proteinase K

• Salting out or precipitation —> Potassium acetate, centrifugation, alc

• Anion exchange —> Backbone bind to pos charged beads

• Cesium chloride gradient —> High purity, time consuming

• Organic solvents (Phenol-Chloroform)

  • Phenol (aqueous)

  • Chloroform (organic)

  • Isoamyl Alcohol

• Alkaline lysis

  • SDS —> breaks phospholipid bilayer

  • NaOH —> dissolves structural proteins

  • Agitation, precip, centrifugation           

• Silica-based —> adsorption of DNA to gels with chaotropic salts

Plasmid Purification

• Cloned genes —> plasmids, NOT DNA

• Miniprep —> 5-50 micrograms of DNA

• Kits —> large plasmids, endotoxin free

DNA Quantification —> determine miniprep’s yield for the following experiments

• A260 of 1.0 = 50 micrograms/mL dsDNA

• A260/A280 > 1.5 = pure

RNase —> ss endoribonuclease

• Fight pathogens + miniprep reagents —> contamination

• Resistant to metal chelating agents

• Survive extreme heat

• Difficult to inactivate, use

  • BME

  • DEPC

  • Guanidium isothiocyanate

  • Recombinant ribonuclease inhibitor

RNA Isolation   

• mRNA composes 1-5% of RNA

• polyA tail binds to oligo-dT beads/columns

RESUME SLIDE 15