BM_Lab10DNAElectrophoresis - Biomed I Laboratory #10...

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Unformatted text preview: Biomed I Laboratory #10 Electrophoresis of DNA Objective To understand the concept and uses of electrophoresis To visualize the difference in migration of supercoiled and linear DNA. To compare the Hind III digested DNA fragments with those predicted in the simulated digestion Structure of DNA DNA is a polymer made of nucleotides or bases Nucleotides units of DNA are joined together by phosphate bonds Phosphate moiety renders a highly negative charge to DNA Nucleotides pair-up to make the double stranded helix Size of DNA is given as "basepairs" (bp) Structure of DNA DNA duplex Supercoiled DNA Negative Backbone Nicked DNA Electrophoresis of DNA DNA molecules migrate from negative to positive electrode Electrophoresis of DNA is an analytical technique used to separate DNA fragments by size. Agarose Gel Agarose is a polysaccharide extracted from seaweed. Agarose polymerizes and crosslinks to form small pores through which DNA can pass through A semi-solid matrix looks much like "Jello". The higher the concentration of agarose, the smaller the pores through which the DNA can move through. For smaller fragments (< 1kb), higher concentration (2%) of agarose is used to get better separation of the DNA fragments Gel Preparation While the solution is still hot, we pour it into a mold called a "casting tray" so it will assume the shape we want as it polymerizes. We use a comb which are removed once the gel solidifies (to create wells) Samples Hind III digested DNA (marker) Hind III digested pUC19 DNA (+ control) Uncut pUC19 DNA (student sample) Hind III digested pUC19 DNA (student sample Sample, Loading Loading buffer contains glycerol and tracking dye To allow the sample to "fall" into the sample wells, To allow visual monitoring or how far the electrophoresis has proceeded. Running gel Add TAE buffer to cover the Gel Wells are near the negative electrode DNA Visualization using SYBR SafeTM The fluorescent dye in SYBR SafeTM intercalates between bases of DNA and RNA. The DNA SYBR complex can absorb UV light and emit it in the visible range. Used to visualize DNA Stain, SYBR SafeTM SYBR SafeTM is incorporated into the gel so that staining occurs during electrophoresis. To visualize DNA or RNA, the gel is placed on a ultraviolet transilluminator. GEL PICS - 1 DNA Uncut Cut pUC19 Uncut Cut Uncut Cut Cut DNA Cut Cut Cut Note: poor resolution Overloading slows DNA improper digestion GEL PICS - 1 DNA Uncut Cut pUC19 pure Uncut Cut unCut Cut StudentpUC19 unCut Cut unCut nicked linear supercoiled RNA ...
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This note was uploaded on 04/24/2008 for the course PHS 2301 taught by Professor Carvalho during the Spring '08 term at St. Johns Duplicate.

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