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A. gel electrophoresis

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gel electrophoresis to separate DNA RNA

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A. gel electrophoresis

  1. 1. Agarose Gel Electrophoresis PRESENTED BY:- AVINASH SHARMA PALB-3235 power supply gel box 26/04/14 PGS 504{0+1} BASIC CONCEPTS IN LABORATORY TECHNIQUES 1
  2. 2. Contents:- Definition of agarose gel electophoresis Procedure of agarose gel electrophoresis Application of gel electrophoresis Problem in agarose gel electrophoresis 26/04/14 PGS 504{0+1} BASIC CONCEPTS IN LABORATORY TECHNIQUES 2
  3. 3. What is agarose? • it is a polymer of D,L galactose and it is a heteropolysaccharide extracted from red algae seaweed. • Low electro endosmosis and good for working. • Agarose has large range of separation but low resolving power. 26/04/14 PGS 504{0+1} BASIC CONCEPTS IN LABORATORY TECHNIQUES 3
  4. 4. Agarose Gel Electrophoresis Gel electrophoresis is a procedure that separates molecules on the basis of size and/or charge and/or shape under the influence of an electrical field. It is based on the principle of “ohms law equation” and”power equation”. Gel electrophoresis is a widely used technique for the separation of nucleic acids and proteins. Agarose gel electrophoresis is routinely used for the separation of DNA. e will be using agarose gel electrophoresis to determine the presence and size of PCR products. +- Power DNA H O2 + 26/04/14 PGS 504{0+1} BASIC CONCEPTS IN LABORATORY TECHNIQUES 4
  5. 5. Procedures of agarose gel electrophoresis:- An agarose gel is prepared by combining agarose powder and a buffer solution. Agarose Buffer Flask for boiling 26/04/14 PGS 504{0+1} BASIC CONCEPTS IN LABORATORY TECHNIQUES 5
  6. 6. Casting tray Gel combs Power supply Gel tank Cover Electrical leads  Electrophoresis Equipment 26/04/14 PGS 504{0+1} BASIC CONCEPTS IN LABORATORY TECHNIQUES 6
  7. 7. Gel casting tray & combs 26/04/14 PGS 504{0+1} BASIC CONCEPTS IN LABORATORY TECHNIQUES 7
  8. 8. Seal the edges of the casting tray and put in the combs. Place the casting tray on a level surface. None of the gel combs should be touching the surface of the casting tray. Preparing the Casting Tray 26/04/14 PGS 504{0+1} BASIC CONCEPTS IN LABORATORY TECHNIQUES 8
  9. 9. Agarose Buffer Solution Combine the agarose powder and buffer solution. Use a flask that is several times larger than the volume of buffer. 26/04/14 PGS 504{0+1} BASIC CONCEPTS IN LABORATORY TECHNIQUES 9
  10. 10. Agarose is insoluble at room temperature (left). The agarose solution is boiled until clear (right). Gently swirl the solution periodically when heating to allow all the grains of agarose to dissolve. ***Be careful when boiling - the agarose solution may become superheated and may boil violently if it has been heated too long in a microwave oven. Melting the Agarose 26/04/14 PGS 504{0+1} BASIC CONCEPTS IN LABORATORY TECHNIQUES 10
  11. 11. Allow the agarose solution to cool slightly (~60ºC) and then carefully pour the melted agarose solution into the casting tray. Avoid air bubbles. Pouring the gel 26/04/14 PGS 504{0+1} BASIC CONCEPTS IN LABORATORY TECHNIQUES 11
  12. 12. Each of the gel combs should be submerged in the melted agarose solution. 26/04/14 PGS 504{0+1} BASIC CONCEPTS IN LABORATORY TECHNIQUES 12
  13. 13. When cooled, the agarose polymerizes, forming a flexible gel. It should appear lighter in color when completely cooled (30-45 minutes). Carefully remove the combs and tape.26/04/14 PGS 504{0+1} BASIC CONCEPTS IN LABORATORY TECHNIQUES 13
  14. 14. Place the gel in the electrophoresis chamber. 26/04/14 PGS 504{0+1} BASIC CONCEPTS IN LABORATORY TECHNIQUES 14
  15. 15. buffer  Add enough electrophoresis buffer to cover the gel to a depth of at least 1 mm. Make sure each well is filled with buffer. Cathode (negative) Anode (positive)  wells    DNA 26/04/14 PGS 504{0+1} BASIC CONCEPTS IN LABORATORY TECHNIQUES 15
  16. 16. Loading Buffer:  • Bromophenol Blue (for color) • Glycerol (for weight) Sample Preparation Mix the samples of DNA with the sample loading buffer ( tracking dye). This allows the samples to be seen when loading onto the gel, and increases the density of the samples, causing them to sink into the gel wells. 26/04/14 PGS 504{0+1} BASIC CONCEPTS IN LABORATORY TECHNIQUES 16
  17. 17. Loading the Gel Carefully place the pipette tip over a well and gently expel the sample. The sample should sink into the well. Be careful not to puncture the gel with the pipette tip. 26/04/14 PGS 504{0+1} BASIC CONCEPTS IN LABORATORY TECHNIQUES 17
  18. 18. Place the cover on the electrophoresis chamber, connecting the electrical leads. Connect the electrical leads to the power supply. Be sure the leads are attached correctly - DNA migrates toward the anode (red). When the power is turned on, bubbles should form on the electrodes in the electrophoresis chamber. Running the Gel 26/04/14 PGS 504{0+1} BASIC CONCEPTS IN LABORATORY TECHNIQUES 18
  19. 19.  wells  Bromophenol Blue Cathode (-) Anode (+) Gel After the current is applied, make sure the Gel is running in the correct direction. Bromophenol blue will run in the same direction as the DNA. DNA (-)  26/04/14 PGS 504{0+1} BASIC CONCEPTS IN LABORATORY TECHNIQUES 19
  20. 20.  100  200  300  1,650  1,000  500  850  650  400  12,000 bp  5,000  2,000 DNA Ladder Standard Inclusion of a DNA ladder (DNAs of know sizes) on the gel makes it easy to determine the sizes of unknown DNAs. - + DNA migration bromophenol blue Note: bromophenol blue migrates at approximately the same rate as a 300 bp DNA molecule 26/04/14 PGS 504{0+1} BASIC CONCEPTS IN LABORATORY TECHNIQUES 20
  21. 21. Staining the Gel ***CAUTION! Ethidium bromide is a powerful mutagen and is moderately toxic. Gloves should be worn at all times. • Ethidium bromide fluorescent dye and binds to the DNA under UV light allowing the visualization of DNA on a Gel. • Ethidium bromide can be added to the gel . 26/04/14 PGS 504{0+1} BASIC CONCEPTS IN LABORATORY TECHNIQUES 21
  22. 22. Staining the Gel • Place the gel in the staining tray containing warm diluted stain. • Allow the gel to stain for 25-30 minutes. • To remove excess stain, allow the gel to destain in water. • Replace water several times for efficient destain. 26/04/14 PGS 504{0+1} BASIC CONCEPTS IN LABORATORY TECHNIQUES 22
  23. 23. Ethidium Bromide requires an ultraviolet light source to visualize 26/04/14 PGS 504{0+1} BASIC CONCEPTS IN LABORATORY TECHNIQUES 23
  24. 24. Factors affecting the DNA in gel elctrophoresis:- • Agarose concentration:- ↑ conc. Of agarose then migration larger fragments of dna and vice versa so for larger fragments of dna agarose conc.=0.7% and smaller fragments of dna agarose conc.=1.5%. • Voltage:- Voltage increases then migration of larger fragments of dna will be more than the smaller dna.so voltage should not be more than 5volts/cm. • Electrophoresis buffer:- TAE and TBE buffers are used in the gel electrophoresis because it establish the pH8.0 and provide ions for the migration of dna and ionic strength are different. • Effects of ethidium bromide:- It is a carcinogenic mutagen and fluorescent dye which are used in the visualizing the dna when agarose gel are placed in the UV transilluminator then dye are free in the gel then dye binds with dna then it produce orange light for visualizing the dna. 26/04/14 PGS 504{0+1} BASIC CONCEPTS IN LABORATORY TECHNIQUES 24
  25. 25. Application of electrophoresis • To separate fragments from each other • To determine the sizes of fragments • To determine the presence or amount of DNA • To analyze restriction digestion products prior to southern transfer or of RNA prior to northern transfer. • Analysis of PCR product. 26/04/14 PGS 504{0+1} BASIC CONCEPTS IN LABORATORY TECHNIQUES 25
  26. 26. Proplems in agarose gel electrophoresis:- • Smearing •voltage too high for large fragments •too much DNA • Gel melts •voltage too high •ionic strength too low • Poor resolution •wrong [agarose] •small bands are fuzzy – the gel run may have been too long at too low a voltage, allowing diffusion of the DNA and broadening of the band 26/04/14 PGS 504{0+1} BASIC CONCEPTS IN LABORATORY TECHNIQUES 26
  27. 27. 26/04/14 PGS 504{0+1} BASIC CONCEPTS IN LABORATORY TECHNIQUES 27 Thank you for your attention

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