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Robin hge

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tools and techniques

tools and techniques

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  • 1. Electrophoresis The Separation of macromolecules under the influence of a uniform electric field through a matrix which is porous in nature is to be termed as ELECTROPHORESIS
  • 2. Electrophoresis Zone Electrophoresis Paper electrophoresis Gel electrophoresis Isoelectric foucusing Immunoelectroph oresis  Different types of electrophoresis
  • 3. • Gel electrophoresis is a method for separation and analysis of macromolecules (DNA, RNA and proteins) and their fragments, based on their size and charge. • Gel electrophoresis is a widely used technique for the analysis of nucleic acids and proteins. • Most every molecular biology research laboratory routinely uses agarose gel electrophoresis for the preparation and analysis of DNA.
  • 4. Agarose Detector
  • 5. •Employs electromotive force to move molecules through a porous gel •Separates molecules from each other on the basis of  size and/or  charge and/or  shape •Basis of separation depends on how the sample and gel are prepared
  • 6. Gel electrophoresis Horizontal gel electrophoresis Vertical gel electrophoresis  Important types of gel electrophoresi
  • 7. Porous Material Proteins Entering Porous Material Smallest Move Fastest •Also called Agarose gel electrophoresis •In this gel electrophoresis the matrix used is a gel and is made up of agarose •Agarose – a complex sugar chain from red seaweed. It has a large pore size good for separating large molecules quickly. Horizontal Gel Electrophoresis
  • 8. Components of an Electrophoresis System Power supply and chamber, a source of negatively charged particles with a cathode and anode Buffer, a fluid mixture of water and ions Agarose gel, a porous material that DNA migrates through Gel casting materials DNA ladder, mixture of DNA fragments of known lengths Loading dye, contains a dense material and allows visualization of DNA migration DNA Stain, allows visualizations of DNA fragments after electrophoresis
  • 9. Buffer Dyes Power Supply + - Agarose gel Cathode Anode
  • 10. Where does the current come from?  A direct current power supply  Ions supplied by the buffer  The charge on the macromolecules being separated  Electrolysis of water
  • 11.  Electrolysis of water  4H2O  2H2 + O2 + 2H2O  self-ionization of water throughout the buffer: 4H20  4H+ + 4OH-  At the negative pole  4H+ + 4e-  2H2  At the positive pole  4OH-  O2 + 2H2O + 4e-
  • 12. What factors affect mobility of linear ds DNA?  Pore size of the gel   [agarose]   pore size   pore size   friction   mobility  Voltage across the gel   voltage   mobility  Length of the DNA molecule  smaller molecules generate less friction and so move faster  Ethidium bromide (stain) intercalated into DNA  decreases charge to mass ratio and so decreases mobility
  • 13. General procedure 1. Casting of gel 2. Loading of gel sample 3. Electrophoresis 4. Staining and visualization 5. Downstream procedure
  • 14. Factors affecting resolution  Resolution = separation of fragments  The “higher” the resolution, the more space between fragments of similar, but different, lengths  Resolution is affected by  agarose type  agarose concentration  salt concentration of buffer or sample  amount of DNA loaded in the sample  voltage
  • 15.  Linear carbohydrate polymer extracted from red seaweed , agarbiose  forms a porous matrix as it gels  shifts from random coil in solution to structure in which chains are bundled into double helices What is Agarose ?
  • 16. % Agarose (w/v) Size Range (kb prs) for Optimal Separation 0.5 2-30 0.75 0.7-20 1.0 0.5-10 1.5 0.2-3 2.0 0.1-2 Resolution of ds linear DNA fragments in agarose gels 1. 1%gels are common for many applications. 2. Up to 3% can be used for separating very tiny fragments but a vertical polyacrylamide gel is more appropriate in this case
  • 17. Buffer Systems  Remember, buffer systems include weak acids and/or bases that do not dissociate completely.  If ions resulting from dissociation are “removed,” more weak acid and/or base will dissociate.  Purposes of buffer  Keep solution at pH compatible with molecules being separated  Generate ions consistently to  maintain current  keep resistance low  Both gel and the solution in the gel box are buffered.
  • 18. Buffer Systems (cont’d)  Two commonly used buffers for routine agarose gel electrophoresis  TAE, pH 8.0, ~50 mM - Tris, Acetate, EDTA  TBE, pH 8.0, ~50 mM - Tris, Borate, EDTA  Tris (T) is a weak base.  Acetic (A) acid and boric (B) acid are weak acids.  Acetic acid is more completely ionized at pH 8.0 than is boric acid, so TBE has a high buffer capacity than TAE.
  • 19. Non-denaturing agarose gel loading solutions  Composition  tracking dyes  are used to follow progress of electrophoresis  sometimes interfere with later visualization of DNA  a solute to increase density  so that sample falls to bottom of loading well with minimal dilution  solute examples: glycerol, Ficoll  Other gel types, with different purposes, use different loading solutions!
  • 20. Voltage   voltage,  rate of migration  to increase the voltage  increase the setting on the power supply  increase the resistance  decrease the gel thickness  decrease the ion concentration  if voltage is too high, gel melts  as voltage is increased, large molecules migrate at a rate proportionally faster than small molecules, so  lower voltages are better for resolving large fragments  but the larger ds DNA fragments are always slower than the smaller ones
  • 21. Ethidium bromide staining  Binds to DNA by intercalation between stacked bases  lies perpendicular to helical axis  makes Van der Waals contacts with bases above and below  Allows DNA visualization after gel electrophoresis  EtBr intercalates with DNA and fluoresce under ultraviolet light thereby allowing DNA visualization after Gel Electrophoresiswhile  Proteins may be visualised using silver stain or Coomassie Brilliant Blue
  • 22.  Agarose Gel Electrophoresis :OVERVIEW