Electrophoresis 1 PDF Lecture Notes

ELECTROPHORESIS 1 By Dr Tsen Min Tze Inspire Empower Elevate  Learning Outcomes Principles of electrophoresis Agarose gel electrophoresis (DNA) Apparatus for agarose gel electrophoresis Capillary electrophoresis Electrophoresis Electrophoresis is a tech...

ELECTROPHORESIS 1 By Dr Tsen Min Tze Inspire Empower Elevate  Learning Outcomes Principles of electrophoresis Agarose gel electrophoresis (DNA) Apparatus for agarose gel electrophoresis Capillary electrophoresis Electrophoresis Electrophoresis is a technique used to separate and sometimes purify charged macromolecules that differ in size, charge and conformation in an electric field. When charged molecules are placed in an electric field, they migrate toward either the positive or negative pole according to their charge.  The greater the charge of a macromolecule, the faster it migrates (greater electrophoretic mobility). Factors Affecting Electrophoresis Greater size of the macromolecule will have greater frictional and electrostatic forces, slower electrophoretic mobility. Rounded molecules have lesser frictional and electrostatic retardation compared to non- globular structure. Globular protein move faster than fibrous protein; supercoil plasmid move faster than linear plasmid. Supercoiled plasmid migrate faster than linearized plasmid. Electrophoresis - Principles Proteins and nucleic acids migrate within a support matrix such as paper, cellulose acetate, starch gel, agarose or polyacrylamide gel. These are solid yet porous matrixes. molecules will move through the matrix at different rates, usually determined by their mass, charge and conformation; they eventually get separated as different ‘bands’. Molecular weight markers are run side by side with samples, the molecular weight of samples can be estimated by referring to the markers. Electrophoresis - Commonly Used Matrixes DNA electrophoresis - Agarose gel. Protein electrophoresis - Polyacrylamide gel.  Agarose Gel Electrophoresis (DNA) Agarose gel is used to separate DNA fragments based on molecular weight. DNA (and RNA) molecules are negatively charged, when placed in an electric field, they migrate to the positively charged anode. DNA fragments (double-stranded linear DNA) has a uniform mass/charge ratio; thus DNA fragments are separated by size within an agarose gel. Shorter fragments migrate faster, longer fragment migrate slower. The distance migrated on gel correlate inversely with the logarithm of molecular weight. How to cast an agarose gel? Gel tray Boil agarose powder in buffer Wait for the gel to set Insert a comb  Loading DNA into wells 1475-9292-5-2-1 Loading Dye While loading the DNA sample into the gel, DNA sample is mixed with loading dye. Loading dye typically contains glycerol (or sucrose) and Bromophenol Blue. Glycerol –high density solution– pull DNA sample and settle to the bottom of the well. Bromophenol Blue – small dye molecules that move through the gel rapidly ahead of all DNA fragments. It is therefore acting as a marker for the electrophoretic front. (Electrophoresis will be stopped before all DNA samples migrate and run out from the end of the gel.) Loading Dye Sample is loaded here. Direction of DNA migration Loading dye pull DNA samples to the bottom of Electrophoretic front the well. DNA Ladder AKA DNA molecular weight marker. It is a set of DNA standards with known base pair size that are used to identify the approximate size of a DNA sample run on electrophoresis. DNA ladder is run side by side with DNA samples. Staining DNA in Agarose Gel DNA are invisible to naked eyes. Ethidium bromide bind to double stranded DNA. Ethidium bromide fluoresce in orange color while irradiated by UV light. Ethidium bromide is normally added when the gel is made; or added to the electrophoresis buffer. After electrophoresis, the gel will be irradiated with UV, image will be taken. Safer alternative: crystal violet, methylene blue, propidium iodate … Staining DNA in Agarose Gel Ethidium bormide bind to Gel image taken and turn double stranded DNA. into black and white image. Ethidium bromide fluoresce when the gel is irradiated with UV light. Capillary Electrophoresis (I hate definition) A method to separate charged molecules by their charge to mass ratio in a capillary tube using electric field and electroosmotic flow. It combines the features of electrophoresis (move charged molecules in an electric field) and chromatography (measure the time required for molecules to travel through a matrix). Automated DNA sequencer. Schematic of capillary electrophoresis system  Electroosmotic Flow Electroosmotic flow (EOF) is the force that moves fluid from one end of a capillary tube to another. The concept of EOF is extremely complex; fortunately, it is possible to carry out capillary electrophoresis without understanding the theory behind it. Electroosmotic Flow Electroosmotic Flow  The inner surface is coated with negatively charged groups. Capillary is filled with buffer and samples. Negative groups attract cations to the inside wall of the capillary, forming a cations double layers: rigid layer and diffuse layer. Diffuse layer is rich in positive charge, but is mobile, not rigidly bound to capillary wall. When a high voltage is applied through the capillary, the cations in the diffuse layer are attracted to the negative electrode, dragging the bulk buffer solution with them (electroosmotic flow). The net result is that this electroosmotic flow will move everything to the negative electrode (cathode). Cations Electrophoretic Mobility + EOF  Fastest Neutral EOF only (not attracted to any electrode) Anions Opposite Electrophoretic Mobility + EOF  Slowest Electroosmotic Flow + When running voltage is too high in electrophoresis, DNA band with turn into parabolic line; due to friction. If the DNA band is driven by electroosmotic pressure, no friction between the band and the capillary tube. The band is uniform across the capillary. - Smiley gel – when voltage applied during electrophoresis is too high. Capillary Electrophoresis – Advantages over Conventional Electrophoresis 1. High speed – because high voltage can be used. 2. High resolution – able to resolve DNA fragments with only 1- bp difference. 3. Yield precise quantitative information – detector to detect the migrated DNA, convert by software into quantitative information. 4. Use only very little sample. THANK YOU