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Gel Electrophoresis 04 How to Prepare the Gel ? 05 Applications of Gel Electrophoresis 01- History

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Gel Electrophoresis 04 How to Prepare the Gel ? 05 Applications of Gel Electrophoresis 01- History Types and applications for gel electrophorasis presented by: samah nasr elshaikh TABLE OF CONTENTS 01 History 02 Introduction 03 Diffreant types of gel electrophoresis 04 How to Prepare the gel ? 05 Applications of gel electrophoresis 01- History First invention invented by Arne Tiselius, a Swedish scientist. •First electrophoresis, moving boundary/ Tiselius electrophoresis. •Then, he invented the new method “zone Arne Tiselius electrophoresis. •Oliver Smithies invented starch gel electrophoresis. Oliver Smithies 02 - introduction where dose the name “ Electrophoresis “ come from ? where dose the name “ Electrophorasis “ come from ? The suffix phoresis from the Greek = to carry across - means "migration" or "movement.“ The prefix electro = flow of electricity , tells us that we are using electricity to make molecules migrate. Then Gel electrophoresis refers to the separation of charged particles located in a gel when an electric current is applied Gel electrophoresis is a technique used to separate DNA fragments (or other macromolecules, such as RNA and proteins) based on their size and charge. Gel 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. What is a gel? As the name suggests, gel electrophoresis involves a gel: a slab of Jello-like material. Gel is a cross linked polymer whose composition and porosity is chosen based on the specific weight of the target molecules . Different types of gels which can be used are agar and agarose gel, starch, sephadex, polyacrylamide gels , cellulose acetate , which comes as dry, powdered flakes. When the powder 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 . How do DNA fragments move through the gel? Once the gel is in the box, each of the DNA samples we want to examine is carefully transferred into one of the wells. One well is reserved for a DNA ladder, a standard reference that contains DNA fragments of known lengths. Commercial DNA ladders come in different size ranges, so we would want to pick one with good "coverage" of the size range of our expected fragments. Next, the power to the gel box is turned on as one end of the gel has a positive charge and the other end has a negative charge. , and current begins to flow through the gel. Because DNA and RNA are negatively charged molecules because of the phosphate groups in their sugar-phosphate backbone, they will be pulled toward the positively charged end of the gel. Proteins, however, are not negatively charged; thus, want to separate proteins using gel electrophoresis, must first mix the proteins with a detergent called sodium dodecyl sulfate ( SDS ). This treatment makes the proteins unfold into a linear shape and coats them with a negative charge, which allows them to migrate toward the positive end of the gel and be separated. Visualizing the DNA fragments Once the fragments have been separated, we can examine the gel and see what sizes of bands are found on it. When a gel is stained with a DNA-binding dye and placed under UV light, the DNA fragments will glow, allowing us to see the DNA present at different locations along the length of the gel. TYPES OF ELECTROPHORESIS 1) Zone Electrophoresis : • Paper Electrophoresis • Gel Electrophoresis • Thin Layer Electrophoresis • Cellulose acetate Electrophoresis 2) Moving Boundary Electrophoresis : • Capillary Electrophoresis • Isotachophoresis • Isoelectric Focussing • Immuno Electrophoresis 03-Different types of gel electrophoresis 1. Agar or Agarose gel electrophoresis : - pulsed field gel electrophoresis For separating larger nucleic acids. 2. Polyacrylamide gel electrophoresis : - native For separating smaller nucleic acids. - SDS-PAGE for denaturing the proteins . 3. starch gel electrophoresis : Non-denatured proteins can be separated according to charge and size . 1- Agarose gel electrophoresis Agar is a mixture of poly saccharides extracted from sea weeds. Agarose is a highly purified uncharged polysaccharide derived from agar. Agarose is chemically basic disaccharide repeating units of 3,6-anhydro-L- galactose Agarose gel is a three-dimensional matrix formed of helical agarose molecules in supercoiled bundles that are aggregated into three- dimensional structures with channels and pores through which biomolecules can pass. The pore size may be predetermined by adjusting the concentration of agarose in the gel the higher the concentration of agarose, the smaller the pore size Gel Structure of Agarose: Traditional agarose gels are most effective at the separation of DNA fragments between 100 bp and 25 kb. To separate DNA fragments larger than 25 kb, one will need to use pulse field gel electrophoresis The 3-D structure is held together with hydrogen bonds and can therefore be disrupted by heating back to a liquid state. The melting temperature is different from the gelling temperature, depending on the sources, agarose gel has a gelling temperature of 35–42 °C and a melting temperature of 85– 95 °C In this method, DNA is forced to migrate through a highly cross-linked agarose matrix in response to an electric current. In solution, the phosphates of the DNA are negatively charged, and the molecule will therefore migrate to the positive electrode. (anode). There are factors that affect migration rate through a gel: 1. size of the DNA 2. agarose concentration 3. voltage applied 4. presence of ethidium bromide 5. conformation of the DNA 6. ionic strength of the running buffer. This matrix creates resistance and means that smaller molecules migrate more quickly while larger molecules migrate more slowly. The difference in migration rate is how we separate the different sizes of DNA molecule to determine their length. 03-Preparation of the Gel Weigh out the appropriate mass of agarose into an 01 Erlenmeyer flask. Agarose gels are prepared using a w/v percentage solution. The concentration of agarose in a gel will depend on the sizes of the DNA fragments to be separated, with most gels ranging between 0.5%- 2%. The volume of the buffer should not be greater than 1/3 of the capacity of the flask. Add running buffer to the agarose-containing flask. Swirl to mix. The most common gel running buffers are TAE (40 mM 02 Tris-acetate, 1 mM EDTA) and TBE (45 mM Tris-borate, 1 mM EDTA). Melt the agarose/buffer mixture. This is most commonly done by heating in a microwave, but can also be done over a Bunsen 03 flame. At 30 s intervals, remove the flask and swirl the contents to mix well. Repeat until the agarose has completely dissolved. Add ethidium bromide (EtBr) to a concentration of 0.5 04 μg/ml. Alternatively, the gel may also be stained after electrophoresis in running buffer containing 0.5 μg/ml EtBr for 15-30 min, followed by destaining in running buffer for an equal length of time. EtBr is a suspected carcinogen and must be properly disposed of per institution regulations. Gloves should always be worn when handling gels containing EtBr. Alternative dyes for the staining of DNA are available however EtBr remains the most popular one due to its sensitivity and cost. Allow the agarose to cool either on the 05 benchtop or by incubation in a 65 °C water bath. Failure to do so will warp the gel tray. Place the gel tray into the casting apparatus. Alternatively, one may also tape 06 the open edges of a gel tray to create a mold. Place an appropriate comb into the gel mold to create the wells. Allow the agarose to set at room temperature. Remove the comb and place the gel in the gel box. Alternatively, the gel can also be 07 wrapped in plastic wrap and stored at 4 °C until use Program the power supply to desired voltage (1-5V/cm between 08 electrodes). Add enough running buffer to cover the surface of the gel. It is important to use the same running buffer as the one used to prepare the gel. Buffers Buffers in gel electrophoresis are used to provide ions that carry a current and to maintain pH at a relatively constant value. Buffers Function Barbitone Buffer( 8.0 pH) Serum protein separation • Poor resolution , weak buffer Phosphate Buffer (pH- 7.5) Enzyme separation • Low buffering capacity :-High conductivity. Tris – borate – EDTA buffer (TBE) Nucleic acid separation • Good resolution , pH- 8.0 high buffering capacity ,low conductivity. Tris – acetate –EDTA buffer (TAE) •Nucleic acid separation • Good resolution , pH – 8.0 high buffering capacity ,low conductivity Tris – glycine buffer (pH8.0) Protein separation • High buffering capacity, low conductivity Add loading dye to the DNA samples to be separated , Gel 09 loading dye is typically made at 6X concentration (0.25% bromophenol blue, 0.25% xylene cyanol, 30% glycerol). Loading dye helps to track how far your DNA sample has traveled, and also allows the sample to sink into the gel. Slowly and carefully load the DNA sample(s) into the gel . 10 An appropriate DNA size marker (ladder) should always be loaded along with experimental samples. Replace the lid to the gel box. The cathode (black leads) should be closer the wells than the anode (red leads). Double check that the 11 electrodes are plugged into the correct slots in the power supply. Turn on the power. Run the gel until the dye has migrated to an appropriate distance. Observing Separated DNA fragments Remove gel from the gel box.
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