2010 Bio 311 Lab C Manual (part 3)

2010 Bio 311 Lab C Manual (part 3) - C3 Plasmid...

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91 C3 . Lab Overview: U sing the alkaline lysis procedure, you will purify the plasmid DNA from the culture that you grew from the selected colony on your master plate. During lab C4, you will analyze the purified plasmid DNA by restriction endonucleases digestion. You will run the uncut DNA on an agarose gel to determine the success of the isolation as well as the size of the plasmid DNA compared to other DNAs. Background Plasmids can be easily isolated from bacteria and purified from bacterial chromosomal DNA and other cellular components using different methods. When volumes of cells of a few milliliters or less are used for preparation of plasmid DNA, it is known as a mini-preparation or “miniprep”. You will be doing a miniprep isolation of plasmid DNA from bacteria you grew last time from your colony PCR inoculations. You will pellet the bacteria and isolate plasmid DNA from the cell pellet. a. Large Scale Preparation of Plasmid DNA The procedure used to isolate plasmid DNA is called the alkaline lysis method. First the cells are removed from the growth media by centrifugation followed by removal of the supernatant and resuspension of the cell pellet in an osmotically favorable buffer. If a large amount of cells (50 to 100 ml) are to be prepared, then the enzyme lysozyme is added to the resuspension buffer. Lysozyme facilitates cell rupture because it cleaves particular carbohydrate linkages that hold together the cell walls of bacteria. This leads to holes in the cell wall. The cell membrane is subsequently solubilized or dissolved with the strong ionic detergent, sodium dodecyl sulfate, SDS, to release the cell contents. In combination with sodium hydroxide, the bacterial chromosomal DNA as well as the plasmid DNA is denaturated and the RNA in the cell partially hydrolyzed (broken up into smaller pieces). This is followed by a neutralization step with an acidic buffer (potassium acetate) to renature the plasmid DNA. The plasmid DNA renatures because it is small (has few base pairs compared to chromosomal DNA). Neutralization leads to the clumping and precipitation of proteins, RNA, chromosomal DNA (too large to properly renature), and the cell membrane. The unwanted precipitate is pelleted by centrifugation and the supernatant containing the plasmid DNA collected. The plasmid DNA in the supernatant is precipitated or removed from ± Read lab C3. ± State lab Goal(s) . ± Outline or make a flow chart of the methods. ± Complete all calculations.
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92 solution by the use of salt and alcohol. The positive ions of the salt form an ionic interaction with the negatively charged phosphates on the DNA. Upon addition of ethanol, the water of hydration is removed from the salt causing it to come out of solution. Since the DNA is in a complex with the salt, it too comes out of solution. The DNA is dried to remove all the ethanol and resuspended in a buffer consisting of 10 mM Tris-HCl, pH 8 1 mM EDTA called “TE”. The high pH from the weak base Tris allows the acidic nucleic acid to dissolve easily.
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This note was uploaded on 03/28/2011 for the course BIO 311 taught by Professor Staff during the Fall '08 term at SUNY Stony Brook.

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2010 Bio 311 Lab C Manual (part 3) - C3 Plasmid...

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