Gel Electrophoresis IB Biology PDF

Introduction of Biotechnology Section 5 Gel Electrophoresis Email ‫قسم الوراثة – كلية الزراعة – جامعة‬ ‫عين شمس‬...

Introduction of Biotechnology Section 5 Gel Electrophoresis Email ‫قسم الوراثة – كلية الزراعة – جامعة‬ ‫عين شمس‬ [email protected]  Outlines 1. What’s Gel 2. How to use it ? Electrophoresis?  What’s Gel Electrophoresis ? Gel electrophoresis is a widely used technique for the analysis of nucleic acids and proteins. This is achieved by moving negatively charged nucleic acid molecules through an agarose matrix with an electric field (electrophoresis).  Gel Electrophoresis Vertical Electrophoresis Horizontal Electrophoresis for proteins for nucleic acids Gel Electrophoresis  Gel Electrophoresis DNA Small Large - - + Power  Gel Electrophoresis Gel electrophoresis separates DNA according to size (Molecular Size) Small DNA move faster than large DNA An agarose gel is used to slow the movement of DNA and separate molecules  Gel Electrophoresis Agarose is a linear polymer extracted from seaweed Gel Electrophoresis  Making an Agarose Gel An agarose gel is prepared by combining agarose powder and a buffer solution  Making an Agarose Gel Agarose is insoluble at room temperature (down) The agarose solution is boiled until clear (up)  Pouring the Gel -Allow the agarose solution to cool slightly (~60ºC) and then carefully pour the melted agarose solution into the casting tray -Avoid air bubbles  Gel Electrophoresis When cooled Agarose polymerizes, forming a flexible gel. It should appear lighter in color when completely cooled (30-45 minutes). Carefully remove the combs and tape Making an Agarose Gel  Gel Electrophoresis Place the gel in the electrophoresis chamber  Gel Electrophoresis Add enough electrophoresis buffer to cover the gel to a depth of at least 1 mm. Make sure each well is filled with buffer.  Running Buffers TAE = Tris Acetic EDTA buffer TBE = Tris Boric EDTA buffer The most common is the second type (TBE), Why? Because it is thermally fixed with high movements, while others are less stable and slimming high temperature.  Visualization Dye EtBr = Ethidium bromide Florescent dye is light purple during exposure to UV. rays, used to see DNA BPB = Bromophenol Blue With samples are placed during the injection into the gel (used as an indicator to move the (DNA)  Bromophenol Blue Dye Mix the samples of DNA with the 6X sample loading buffer (w/ 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. 6X Loading Buffer: - Bromophenol Blue (for color) - Glycerol (for weight)  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.  Ethidium Bromide Dye Ethidium bromide binds to **CAUTION! Ethidium bromide is a DNA and fluoresces under powerful mutagen and is moderately U.V. light, allowing the toxic. visualization of DNA on a Gel. Gloves should be always worn.  Running the Gel 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.  Migration of DNA Cathode (-)  wells DNA  Bromophenol Blue (-)  Gel Anode (+) 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. UV. Trans-illuminator UV. Trans-illuminator U.V. Trans-illuminator Gel Electrophoresis  Final Agarose gel Figure: Agarose gel electrophoresis of DNA purified with SpinClean™ Genomic DNA purification Kit. M: 1 kb ladder marker line 1: Chicken whole blood (20 µl sample volume) line 2: Human whole blood(100 µl sample volume) line 3: E. coli. line 4: L. brevis. line 5: Streptomyces sp.  References https://vlab.amrita.edu/?sub=3&brch=77&sim=1375&cnt=2 https://learn.genetics.utah.edu/content/labs/gel/ https://vimeo.com/343712593 https://worldwide.promega.com/resources/product-guides-and-selectors/protocols-and-applications-guide/pcr-amplification/ https://www.britannica.com/science/polymerase-chain-reaction Do you have any Question ? ‫قسم الوراثة – كلية الزراعة – جامعة عين شمس‬ Email [email protected]  How to Contact Us Facebook www.facebook.com/-‫شمس‬-‫عين‬-‫جامعة‬-‫الزراعة‬-‫كلية‬-‫الوراثة‬-‫قسم‬ 101746015601374 Twitter https://twitter.com/Genetics_Shams Telegram https://t.me/GeneticsAgrASU Website GeneticsASU.blogspot.com Email ‫قسم الوراثة – كلية الزراعة – جامعة عين شمس‬ [email protected]