Robin hge


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Robin hge

  1. 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. 2. Electrophoresis Zone Electrophoresis Paper electrophoresis Gel electrophoresis Isoelectric foucusing Immunoelectroph oresis  Different types of electrophoresis
  3. 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. 4. Agarose Detector
  5. 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. 6. Gel electrophoresis Horizontal gel electrophoresis Vertical gel electrophoresis  Important types of gel electrophoresi
  7. 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. 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. 9. Buffer Dyes Power Supply + - Agarose gel Cathode Anode
  10. 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. 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. 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. 13. General procedure 1. Casting of gel 2. Loading of gel sample 3. Electrophoresis 4. Staining and visualization 5. Downstream procedure
  14. 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. 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. 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. 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. 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. 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. 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. 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. 22.  Agarose Gel Electrophoresis :OVERVIEW