Successfully reported this slideshow.
We use your LinkedIn profile and activity data to personalize ads and to show you more relevant ads. You can change your ad preferences anytime.

Gel electrophorosis final


Published on

Published in: Technology
  • Be the first to comment

Gel electrophorosis final

  1. 1. Gel Electrophoresis NAYANA.P and Jitendra Kumar Dept OF FRM COLLEGE OF FISHERIES
  2. 2. Introduction Gel electrophoresis is a method used in clinical chemistry to separate proteins by charge and or size and in biochemistry and molecular biology to separate a mixed population of DNA and RNA fragments by length, to estimate the size of DNA and RNA fragments or to separate proteins by charge. Nucleic acid molecules are separated by applying an electric field to move the negatively charged molecules through an agarose matrix.
  3. 3. Gel electrophoresis separates molecules on the basis of their charge and size. The charged macromolecules migrate across a span of gel because they are placed in an electrical field. The gel acts as a sieve to retard the passage of molecules according to their size and shape. Shorter molecules move faster and migrate farther than longer ones because shorter molecules migrate more easily through the pores of the gel. This phenomenon is called sieving
  4. 4. Gel electrophoresis is usually performed for analytical purposes, often after amplification of DNA via PCR, but may be used as a preparative technique prior to use of other methods such as mass spectrometry, RFLP,  PCR, cloning, DNA sequencing, or  Southern blotting for further characterization.
  5. 5. Types of gel Agarose Polyacrylamide Starch
  6. 6. Agarose  gels are easily cast and handled compared to other matrices, because the gel setting is a physical rather than chemical change. Samples are also easily recovered. After the experiment is finished, the resulting gel can be stored in a plastic bag in a refrigerator. advantages: it is used for the separation of DNA fragments ranging from 50 base pair to several millions of bases using specialized apparatus. The distance between DNA bands of a given length is determined by the percent agarose in the gel
  7. 7. The disadvantage of higher concentrations is the long run times (sometimes days). Low percentage gels are very weak and may break when you try to lift them. High percentage gels are often brittle and do not set evenly Agarose gels do not have a uniform pore size, but are optimal for electrophoresis of proteins that are larger
  8. 8. Polyacrylamide Polyacrylamide gel electrophoresis (PAGE) is used for separating proteins Pore size is controlled by controlling the concentrations of acrylamide and bisacrylamide powder used in creating a gel.
  9. 9. Starch Partially hydrolyzed potato starch makes for another non-toxic medium for protein electrophoresis. The gels are slightly more opaque than acrylamide or agarose. Non-denatured proteins can be separated according to charge and size. They are visualized using Napthal Black or Amido Black staining.
  10. 10. How does it work? • DNA is cut into smaller fragments. • Loading dye is used to indicate the fragments of DNA are behind the dye • The negative DNA molecule is attracted to the positive electrode. • The smallest fragments move the greatest distance.
  11. 11. Gel electrophoresis A method of separating DNA in a gelatin-like material using an electrical field ◦ DNA is negatively charged ◦ when it’s in an electrical field it moves toward the positive side DNA – →→→→→→→ “swimming through Jello” +
  12. 12. Gel electrophoresis DNA moves in an electrical field… ◦ so how does that help you compare DNA fragments?  size of DNA fragment affects how far it travels  small pieces travel farther  large pieces travel slower & lag behind DNA – →→→→→→→ “swimming through Jello” +
  13. 13. Gel Electrophoresis DNA & restriction enzyme longer fragments wells power source gel shorter fragments + completed gel
  14. 14. fragments of DNA separate out based on size Running a gel cut DNA with restriction enzymes 1 2 3 Stain DNA ◦ ethidium bromide binds to DNA ◦ fluoresces under UV light
  15. 15. Procedure Remove comb and observe wells. Place carbon paper in each end of the tray. Cover with buffer, making sure the allow buffer to overflow into each end of the tray. Load gels. Connect the electrodes. Turn on power supply. Allow gels to run – make sure you see bubbles coming from the electrodes.
  16. 16. PROCEDURE (CONTINUED) It will take about 30 minutes for the gel to run. Turn off power supply and remove electrodes. Pour off buffer into the designated container. Carefully remove gel from gel box and place in glad container and cover with stain. Store in appropriate location.
  17. 17. Applications Estimation of the size of DNA molecules following restriction enzyme digestion, e.g. in  restriction mapping of cloned DNA. Analysis of PCR products, e.g. in molecular genetic diagnosis or genetic fingerprinting Separation of restricted genomic DNA Gel electrophoresis is used in forensics, molecular biology, genetics, microbiology and biochemist ry.
  18. 18. Proteins can also be run on gels. Most commonly proteins are run on gels made of polyacrylamide in the presence of SDS Scientific dyes can also be separated by gel electrophoresis.
  19. 19.