GEL ELECTROPHORESIS

In this slide contains principle, types, materials used, factors affecting gel electrophoresis.
Presented by: I. Sai Reddemma (Department of pharmacology).
RIPER, anantapur.

1. 1 A Seminar as a part of curricular requirement for I year M. Pharm I semester Presented by Ms. I. Sai Reddemma. (Reg. No. 20L81S0101) Department of Pharmacology Under the guidance/Mentorship of Dr. P. Ramalingam., Ph.D. Director- R&D Division, Professor of Pharmaceutical Analysis and Medicinal Chemistry. GEL ELECTROPHORESIS

2. 2 Contents Introduction Principle Types of Electrophoresis Materials required for Gel Electrophoresis Factors affect Gel Electrophoresis Procedure of Gel Electrophoresis Applications References

3. 3 Electrophoresis is the migration of charged particles or molecules in a medium under the influence of an electric field. DNA – DNA Gel Electrophoresis (Agarose gel electrophoresis). PROTEINS – SDS PAGE (Sodium Dodecyl Sulphate Polyacrylamide Gel Electrophoresis). Introduction

4. 4 Mixture of Charged Molecules Charge S e p arat i on Negative M o l e c u l e s Positive Molecules Size S e p arat i on

5. 5 A separation technique. Simple, rapid and highly sensitive. Used in clinical laboratories to separate charged molecules from each other in the presence of electric field. To separate DNA, RNA, Proteins (based on Charge and Size). To determine the sizes of DNA fragments. Electrophoresis :

6. 6 Comprehensive term that refers to the migration of charged particles of any size in liquid media under the influence of an electric field. Depending on kind of charge the molecule carry, they move towards either To Cathode Or to Anode An ampholyte become positively charged in acidic condition and migrate to Cathode, in alkaline condition they become negatively charge and migrate to Anode. Shorter molecules move or migrate faster than longer ones . Principle

7. 7 Zone Electrophoresis A. Gel Electrophoresis B. Paper Electrophoresis C. Thin layer Electrophoresis D. Cellulose acetate Electrophoresis 2 . Moving Boundary Electrophoresis A. Isotachophoresis B. Capillary Electrophoresis C. Isoelectric Focussing D. Immuno Electrophoresis Types of Electrophoresis

8. 8 Electrophoresis chamber Agarose gel Gel casting tray Buffer Staining agent (dye) Comb DNA ladder Materials required for Gel Electrophoresis

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10. 10 Preparations of ethidium bromide (Dye) Weigh 10 mg ethidium bromide into a sterile tube and dissolve in 10 ml sterile distilled water. The stock is stored at 4 °C . Preparation of agarose solution for casting the gel Dissolve the Agarose by placing the flasks in boiling water both cool to Luke warm. Cover the sides of a tray using cellotape and place the comb about 1 cm from the top of the tray. Pour the Agarose with out making any bubles, cool it for 20 mins and take off the combs and uncover the cellotapes Preparation of stock solutions

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13. 13 1. Agarose concentration Higher concentration of gels are used for the separation of lower molecular weight DNA and RNA fragments and vice-versa. 2. Molecular weight DNA fragment migrates at rates inversely proportional to the log molecular weight. A plot of logM.W vs. Mobility gives a straight line Factors affect the rate of migration of nucleic acids

14. 14 3. Applied Voltage At low voltage (<5V/cm) the rate of migration is directly proportional to the applied voltage. However, if the voltage is increased, mobility of high molecular weight DNA fragments increased differentially. 4. Buffer I t provides the necessary ion to conduct electricity . H elps maintain a stable p H and a stable temperature. A buffer also keeps the gel from melting.

15. 15 5. Charge Higher the charge greater the electrophoresis mobility. 6. Shape Rounded contours elicit lesser frictional and electrostatic retardation compared to sharp contours. Therefore, globular protein moves faster than fibrous protein.

16. 16 STEP 1 : An Agarose and buffer solution is poured into a tray and chamber. A Comb is placed into the tray on one end Procedure of Gel Electrophoresis

17. 17 STEP 2 : The Agarose polymerizes into a gel as it cools. The Comb is removed from the gel to form Wells for samples.

18. 18 STEP 3 : DNA Samples coloured with a tracking dye are pipette into the Wells.

19. 19 STEP 4 : The tray is placed in a chamber that generates electric current through the gel. The negative electrode is placed on the nearest side of the samples. The positive electrode is placed on other side.

20. 20 STEP 5 : DNA has a negative change and move towards the positive electrode. Smaller DNA molecules will be able to move faster than longer molecules through the gel.

21. 21 STEP 6 : One well, called a DNA ladder will contain DNA fragments of known sizes. This ladder is used to determine the sizes of other samples.

22. 22 An overview of Gel Electrophoresis

23. 23 In the separation of DNA fragments for DNA fingerprinting to investigate crime scenes . To analyze results of polymerase chain reaction . To analyze genes associated with a particular illness . In DNA profiling for taxonomy studies to distinguish different species . In paternity testing using DNA fingerprinting . In the study of structure and function of proteins . In the analysis of antibiotic resistance . In blotting techniques for analysis of macromolecules . Applications of Electrophoresis

24. 24 Gordon, A. H. Electrophoresis of proteins in polyacrylamide and starch gels. American Elsevier Publishing Company. Inc. New York.1975. Smisek , D. L, Hoagland, D. A. Agarose gel electrophoress of high molecularweight,syntheticpolyelectrolytesMacromolecules.22(5):1989,p.2270-2277. Lodish , H, Berk, A, Matsudaira , P. Molecular Cell Biology.5 th edition, WH Freeman,New York.2004. References

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