BCHM 3050 1st Edition Lecture 17 Outline of Last Lecture I Information Processing II Cruciform DNA III EcoR1 Restriction Site IV DNA Gel Electrophoresis V Sanger Sequencing Outline of Current Lecture I How Sanger Sequencing Works II Southern Blotting Current Lecture I How Sanger Sequencing Works a Based on sticky ends you can introduce foreign DNA into a piece of existing DNA b DNA strands are complementary to each other c Phosphodiester bonds exist between nucleotides need an OH group at the 3 end d Sanger generated di deoxynucleotides ddGtP ddCTP ddTTP ddATP e He replaced the OH with just an H which cannot form a phosphodiester bond cannot extended the DNA chain f Has 4 tubes g 1 adds 1 strand of DNA dATP CTP GTP TTP ddATP h 2 adds 1 strand of DNA dATP CTP GTP TTP ddGTP i 3 adds 1 strand of DNA dATP CTP GTP TTP ddCTP These notes represent a detailed interpretation of the professor s lecture GradeBuddy is best used as a supplement to your own notes not as a substitute j 4 adds 1 strand of DNA dATP CTP GTP TTP ddTTP k These di deoxynucleotides prevent the DNA chains from being able to extend l Radioactively labeled the phosphate groups of the dd versions of the nucleotides m He is trying to recreate the bottom strand of the original DNA n Read 5 to 3 bottom to top o Color code nucleotides by measuring the intensity of the color can determine the sequence II Southern Blotting a Much less used procedure b Uses restriction enzymes to cut up large strand of DNA into tiny pieces c Two pieces of DNA that he is interested in are darkly shaded d Runs all pieces of DNA in agrose gel e Figures out where DNA of interest is by sending a probe which is the actually sequence of DNA labels it and adds it to the DNA gel f Can also put filter paper on top of gel and filter paper will absorb all pieces of DNA through adhesion g Send radioactive probe and whereever the DNA of interest is the probe will light up h Southern Blotting isnt very helped but can tell you if your DNA of interest is present doesn t tell you exactly where to find it
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