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Gel electrophoresis
- 2. Self Intro! FATIMA ASLAM KHAN PL01 fatimaaslamkhann@gmail.com
- 5. Electrophoresis • Electro = flow of electricity, • Phoresis = to carry across • Electrophoresis is a technique commonly used in the lab to separate charged molecules, like DNA, according to size. • Is a separation method that is based on the migration of charged species in a supporting medium (a liquid or a hydrophilic gel) under the influence of an electric field
- 6. Principle • An electrophoretic system consists of two electrodes of opposite charge (anode, cathode), connected by a conducting medium called an electrolyte. • The separation effect on the ionic particles results from differences in their velocity (v), which is the product of the particle's mobility (m) and the field strength (E): V=Me
- 8. Zone Electrophoresis Paper Electrophoresis Gel Electro phoresis Thin Layer Electrophor esis Cellulose Bounding Electrophoresis 01 02 03 04
- 10. Gel Electrophoresis Electrophoresis involves running a current through a gel containing the molecules of interest. Based on their size and charge, the molecules will travel through the gel in different directions or at different speeds, allowing them to be separated from one another. Gel electrophoresis is a technique used to separate DNA fragments (or other macromolecules, such as RNA and proteins) based on their size and charge. What's it? How?
- 11. What is a Gel? ● Gel electrophoresis involves a gel: a slab of Jello-like material. ● Gel is a colloid in a solid form (99% is water). ● Gels for DNA separation are often made out of a polysaccharide called agarose, which comes as dry, powdered flakes. ● When the agarose is heated in a buffer (water with some salts in it) and allowed to cool, it will form a solid, slightly squishy gel. ● At the molecular level, the gel is a matrix of agarose molecules that are held together by hydrogen bonds and form tiny pores. At one end, the gel has pocket-like indentations called wells, which are where the DNA samples will be placed. ● Before the DNA samples are added, the gel must be placed in a gel box.
- 12. What is a Gel?
- 13. What is a Gel? ● One end of the box is hooked to a positive electrode, while the other end is hooked to a negative electrode. ● The main body of the box, where the gel is placed, is filled with a salt-containing buffer solution that can conduct current.The buffer fills the gel box to a level where it just barely covers the gel. ● The end of the gel with the wells is positioned towards the negative electrode. ● The end without wells (towards which the DNA fragments will migrate) is positioned towards the positive electrode..
- 14. Types of Gels Matrix Used Poly acryl amide Agarose 01 02 • Large pore size gel will be made • Gel casted horizontally • Used to separate DNA , RNA • Staining can be done before pouring the gel • Small pore size gel will be made • Gel casted vertically • Used to separate Protein & Small size DNA >1000 bp • Staining can be done after pouring the gel
- 15. Agarose Gels • Concentration – 1% - 3% • Separation in agarose gels is achieved due to size (MW) of DNA because of resistance to their movement caused by the gel matrix • Agarose dissolves when added to boiling liquid and forms gels when temperature is lowered to 40°C • Staining is done by ethidium bromide SYBR green and methylene blue • Agarose is a linear polysaccharide : Agarobiose comprises alternating units of galactose and 1,3- anhydrogalactose • Commomly used,less expensive,provides good separation What's it? How Much?
- 17. Poly acryl amide Gels • It is prepared by polymerizing acryl amide monomers in the presence of methylene-bis-acrylamide to cross link the monomers. • Structure of acrylamide (CH=CH-CO-NH) • Polyacrylamide gel structure held together by covalent cross-links. • Polyacrylamide gels are tougher than agarose gels. 'It is thermostable, transparent, strong and relatively chemically inert. • Gels are uncharged and are prepared in a variety of pore sizes. • Proteins are separated on the basis of charge to mass ratio and molecular size
- 18. Buffer Used • Buffers are said to be a mixture of weak acid and its conjugate base as well as vice versa. • As buffers, they also have a fairly constant pH & are able to conduct electricity because of their concentration of hydrogen ions What's it?
- 19. Buffer Used • Tris acetate EDTA (TAE) and tris borate EDTA (TBE) are two most common running buffers used in nucleic acid electrophoresis. • Agarose gel electrophoresis of the DNA and RNA is routinely performed using the buffers containing either Tris, acetate and EDTA (TAE) or Tris, borate and EDTA (TBE). • TAE also works better for cloning, because TBE contains borate. Borate in TBE is an inhibitor for many kind of enzymes, such as ligase. TAE works better for performing the DNA extraction from agarose gel. Types of Buffers
- 20. Stains Used • Ethidium bromide (EtBr) is used for DNA staning to observe DNA in a gel. EtBr binds between the double helix and gives bright orange colour when observed under UV light. • The separated fragments in a mixture can be visualized by ethidium bromide staining followed by UV exposure. Ethidium bromide is added in the agarose gel once agarose powder is dissolved in buffer and mixed thoroughly. • The stain gets dispersed throughout the gel. Ethidium bromide acts as an intercalating molecule. • It gets inserted within the species present between the double helix of DNA. • This absorbs wavelength corresponding to ultraviolet radiation and emits another wavelength that is visible to us.
- 21. Stains Used
- 22. • DNA is an organic acid, and is negatively charged (remember, DNA for Negative) • When the DNA is exposed to an electrical field, the particles migrate toward the positive electrode • Smaller pieces of DNA can travel further in a given time than larger pieces Working How do DNA fragments move through the gel?
- 23. Steps in running a gel How do DNA fragments move through the gel? • DNA is prepared by digestion with restriction enzymes • The gel chamber is set up, the 'comb' is inserted • The agarose may have a DNA 'dye' added (or it may be stained later). The agarose is poured onto the gel block and cooled • The comb is removed, leaving little 'wells' and buffer is poured over the gel to cover it completely • The DNA samples are mixed with a dense loading dye so they sink into their wells and can be seen
- 24. Steps in running a gel How do DNA fragments move through the gel? • The DNA samples are put in the wells with a micropipette(Micropipettes have disposable tips and can accurately measure 1/1,000,000 of a litre) • The power source is turned on and the gel is run. • The time of the run depends upon the amount of current and % gel • At the end of the run the gel is removed (it is actually quite stiff) • The gel is then visualized • UV light causes the bands of DNA to fluoresce • The DNA band of interest can be cut out of the gel and the DNA extracted -Or DNA can be removed from the gel by Southern Blotting
- 26. Advantages of Agarose Gel Electrophoresis • Easy to prepare and small concentration of agar is required. • Resolution is superior to that of filter paper. • Large quantities of proteins can be separated and recovered. • Adsorption of negatively charged protein molecule is Negligible. • It adsorbs proteins relatively less when compared to other medium. • Sharp zones are obtained due to less adsorption. • Recovery of protein is good, good method for preparative purpose
- 27. Disadvantages of Agarose Gel Electrophoresis • Electro osmosis is high. • Resolution is less compared to polyacrylamide gels. • Different sources and batches of agar tend to give different results and purification is often necessary.
- 28. Applications • Widely used in Immuno electrophoresis. • To separate different types of protein mixtures as well as nucleic acids. • Used for estimation of molecular weight of proteins and nucleic acids. • Determination of subunit structure of proteins. • Purification of isolated proteins. • Monitoring changes of protein content in body fluids. • Identifying disulfide bonds between protein • Quantifying proteins • Blotting applications
- 29. DNA Electrophoresis Video Link DNA Electrophoresis