Anam siddique gel electrophoresis

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Gel electrophoresis methodology step wise.. …

Gel electrophoresis methodology step wise..
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  • 1. Presented by : Anam Siddique
  • 2. Agarose Gel electrophoresis and Southern blotting 1/24/2014 8:44:28 AM 2
  • 3. • Contents Introduction of electrophoresis Types of gel electrophoresis Principle Procedure Applications Advantages & disadvantages Southern blotting References 1/24/2014 8:44:28 AM
  • 4. Introducti on 1/24/2014 8:44:28 AM
  • 5. Electrophor esis “The movement of suspended particles through a medium under the action of an electromotive force applied to electrodes in 1/24/2014 8:44:28 AM contact with the
  • 6. Gel electrophoresis A gel is a colloid, a suspension of tiny particles in a medium, occurring in a solid form (like gelatin). 1/24/2014 8:44:28 AM
  • 7. Gel electrophoresis refers to the separation of charged particles located in a gel when an electric current is applied. Charged particles can include DNA, amino acids, 1/24/2014 8:44:28 AM polypeptides, etc. 7
  • 8. Types of gels used in electrophoresis 1/24/2014 8:44:28 AM
  • 9. Gel Agarose 1/24/2014 8:44:28 AM Polyacrylamide
  • 10. Agarose Polysaccharide Used at concentrations of 0.5 to 2% The higher the agarose concentration the "stiffer" the gel Used for the separation of 1/24/2014 8:44:28 AM DNA fragments
  • 11. Polyacrylamide Polyacrylamide gel electrophoresis (PAGE) Used to separate proteins It is non toxic however un polymerized acrylamide is neurotoxin 1/24/2014 8:44:28 AM
  • 12. Agarose Larger 'pores' Large particles can move more easily Used for the electrophoresis of large molecules such as DNA and RNA Agarose gels have a large range of separation, but relatively low resolving power 1/24/2014 8:44:28 AM Polyacrylamide Small pores Large particles cannot move easily Used for electrophoresis of small molecules such as proteins Polyacrylamide gels have a rather small range of separation, but very high resolving power
  • 13. Principle 1/24/2014 8:44:28 AM
  • 14. The agarose gel after solidification has some gaps called PORES. These pores are the channels through which charged molecule has to travel. When charged molecules are placed in an electric field, they migrate toward either the positive or negative pole according to their charge. Nucleic acids have negative charge due to their phosphate backbone. The migration flow is determined solely by the molecular weight. 1/24/2014 small weight molecules migrate faster 8:44:28 AM than larger ones.
  • 15. DNA buffer   wells Cathode (negative) 1/24/2014 8:44:28 AM Anode (positive)
  • 16. All bands are DNA fragments Band 1 longest fragment so nearest to well It moved slowest. Band 10 is shortest and moved fast. 1/24/2014 8:44:28 AM
  • 17. Method 1/24/2014 8:44:28 AM
  • 18. Materials required DNA sample Agarose Ethidium bromide 6X sample loading dye DNA ladder standard Power supply Gel casting tray & comb Pipette and tips Electrophoresis chamber Buffers 1/24/2014 Transilluminator 8:44:28 AM The movement of suspended particles through a medium under the action of an electromotive force applied to electrodes in contact with the suspension”
  • 19. 1. Dissolve 1/24/2014 8:44:28 AM agar in buffer
  • 20. 2. Add ethidium bromide (1µl) in the agarose gel. 1/24/2014 8:44:28 AM
  • 21. 3. Pour the gel in casting tray 1/24/2014 8:44:28 AM
  • 22. 4. Insert the comb and let the gel solidify 1/24/2014 8:44:28 AM
  • 23. 5. After solidification of gel, carefully remove the comb and place the gel in the gel rig with the wells closest to the cathode (black) end. Cover the gel with 1X TAE running buffer. 1/24/2014 8:44:28 AM
  • 24. 6. Keeping DNA samples on ice, pipette up 5 µl of a sample, add it to one drop (1μl) of loading dye. 1/24/2014 8:44:28 AM
  • 25. 7. Load the mixture into a well 1/24/2014 8:44:28 AM
  • 26. 8. Place the cover on the gel rig and run the samples towards the anode (red) end. And run it at 120 volts for about half an hour. 1/24/2014 8:44:28 AM
  • 27. 9. Turn off the power pack, remove the gel and visualize with transilluminator or U.V. light. 1/24/2014 8:44:28 AM
  • 28. 10. Take a photograph with a polaroid Photo documentation camera. 1/24/2014 8:44:28 AM
  • 29. Functions of materials 1/24/2014 8:44:28 AM
  • 30. Ethidium bromide is used for staining It will bind with DNA and & it will emitt fluoresence mutagenic and should be handled with extreme caution 1/24/2014 8:44:28 AM
  • 31. Loading dye Gives colour and density to the sample the dyes are negatively charged and thus move in the same direction as the DNA Bromophenol blue for dying Glycerol for density 1/24/2014 8:44:28 AM
  • 32. DNA ladder standard useful for quantifying the amount of DNA in your sample bands by comparison with the marker bands 1/24/2014 8:44:28 AM
  • 33. Tank buffers TAE buffer (Tris Acetate EDTA) TBE buffer (Tris borate buffer) sequesters divalent cations 1/24/2014 8:44:28 AM
  • 34. TAE buffer TAE offers the best resolution for larger DNA TAE requires a lower voltage and more time linear, double stranded DNA runs faster in TAE. 1/24/2014 8:44:28 AM
  • 35. 1X TAE Buffer: 1. Trisma base = 242g 2. Glacial acetic acid = 57.1ml 3. 0.5M EDTA = 100ml Mix all the constituents and raise the 1/24/2014 volume up to 1L with distilled 8:44:28 AM
  • 36. TBE buffer TBE buffer (Tris/Borate/EDTA) is often used for smaller DNA fragments (ie less than 500bp) 1/24/2014 8:44:28 AM
  • 37. Visualizing DNA 1/24/2014 8:44:28 AM
  • 38. Ethidium Bromide binds to DNA Fluorescence under UV light makes bands visible. 1/24/2014 8:44:28 AM
  • 39. Results 1/24/2014 8:44:28 AM
  • 40. Unique bands and or banding patterns. Molecular weight standards can be ran with samples to estimate sizes.. Migration or separation of a sample can be measured (Rf values). 1/24/2014 8:44:28 AM
  • 41. Factors affecting Gel electrophoresi s 1/24/2014 8:44:28 AM
  • 42. Voltage used Running time Amount of DNA used Reversal of polarity 1/24/2014 8:44:28 AM
  • 43. Applications 1/24/2014 8:44:28 AM
  • 44. It can be used to determine the sequence of nitrogen bases. the size of an insertion or deletion the presence of a point mutation to distinguish between variable sized alleles at a single locus to assess the quality and quantity of DNA present in a sample DNA Sequencing 1/24/2014 8:44:28 AM
  • 45. Blotting (Southern Blot) Size of DNA can be determined by comparing it with ladder. Medical Research Forensics (Criminal investigations) like Blood, Saliva, Hair, nail Paternity Tests 1/24/2014 8:44:28 AM
  • 46. Advantages and disadvantages 1/24/2014 8:44:28 AM
  • 47. Advantages Cheap Quick Easy Sample recovery 1/24/2014 8:44:28 AM
  • 48. Disadvantage Toxicity DNA visualization uses dyes like Ethidium Bromide which chelate DNA and is a known carcinogen. Although some safer alternatives are becoming available (SYBR Safe). • 1/24/2014 8:44:28 AM
  • 49. Southern Blotting 1/24/2014 8:44:28 AM
  • 50. Definition A technique which allows the detection of a specific DNA sequence (gene or other) in a large, complex sample of DNA. 1/24/2014 8:44:28 AM
  • 51. Principle Southern blotting combines agarose gel electrophoresis for size separation of DNA with methods to transfer the size separated DNA to a filter membrane for probe 1/24/2014 hybridization. 8:44:28 AM
  • 52. Procedure Digestion + gel electrophoresis DNA is transferred to a sheet of special blotting paper Label with specific DNA probe Detected on the Xray film 1/24/2014 8:44:28 AM
  • 53. 1/24/2014 8:44:28 AM
  • 54. Applications To identify a single gene among thousands of fragments of DNA and to detect sequences of DNA in an organism’s genome. Used in gene discovery and gene mapping. To analyze the genetic patterns in an organism’s DNA. To identify gene mutations, deletions, duplications, a nd gene rearrangements involved in diseases. 1/24/2014 To determine the number of copies 8:44:28 AM
  • 55. To identify related DNA sequences in the genome and to determine if there is a gene family (a group of similar genes. To detect certain cancers and genetic diseases, such as: Monoclonal leukemia populations Sickle cell mutations Used in DNA fingerprinting, genetic engineering, & forensic science for tests such as Paternity testing Sex determination 1/24/2014 8:44:28 AM
  • 56. References 1/24/2014 8:44:28 AM
  • 57. • • • • • • • • http://en.wikipedia.org/wiki/Gel_electrophoresis http://www.methodbook.net/dna/agarogel.html http://abe.leeward.hawaii.edu/Protocols/DNA%20Gel%20Prep aration%20and%20Electrophoresis.htm http://arbl.cvmbs.colostate.edu/hbooks/genetics/biotech/gels/ principles.html http://www.academia.edu/1546698/Gel_Electrophoresis__Principles_and_Basics www.biotecharticles.com/.../Agarose-Gel-DNA-Electrophoresis Applications www.molecularstation.com/agarose-gel-electrophoresis http://biology.arizona.edu/sciconn/lessons2/vuturo/vuturo/gel .htm 1/24/2014 8:44:28 AM
  • 58. Animations • http://www.dnalc.org/resources/animatio ns/gelelectrophoresis.html • http://learn.genetics.utah.edu/content/lab s/gel/ • http://highered.mcgrawhill.com/sites/0072 556781/student_view0/chapter14/animati on_quiz_5.html 1/24/2014 8:44:28 AM
  • 59. 1/24/2014 8:44:28 AM Agarose Gel electrophoresis and southern blotting 59
  • 60. 1/24/2014 8:44:28 AM