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04b_pDNA PREP_100109 - Purification of Plasmid DNA(pDNA...

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Purification of Plasmid DNA (pDNA) Using The Alkaline Lysis Miniprep Procedure LABORATORY EXERCISE #4 Rev: 3/10/10 Principle(s) of the Procedure - Strategies for the isolation of pDNA take advantage of the physical differences between linear, closed, and supercoiled DNA. - After denaturation at an alkaline pH, closed DNA will quickly renature upon neturalization in the cold. - Conversely, long gDNA will not renature, will remain “tangled” with proteins, SDS, and lipids, and therefore will precipitate (“salting-out”). 1) Inoculate 1.5 mL of LB broth with the isolate and incubate for 18-36 hr at 30-37 ° C - 1
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Performed step last week and stored at 4 ° C since 2) Harvest the cells that were cultured last week - Sediment the cells by centrifugation using a microcentrifuge [12,000 rpm for 3 min] - Being careful not to disturb the cell pellet, pour off and discard the supernatant, remove the remaining fluid using a pipette gun, - Save the pellet , the small off-white smear located at the tube’s side/bottom beneath the hinge 3) Suspend the Cells in 100 μ L of ice-cold GTE* - " BUFFER 1 " - COMPLETELTY Suspend the cell pellet * 50 mM Glucose, 25 mM Tris-HCL (pH 8), 10 mM EDTA - The glucose will “buffer” the effects of the NaOH in Buffer 2 - The EDTA aids in degradation of the cell wall (especially if lysozyme is added to the GTE) 4) Incubate the tube at room temperature for 5 min 5) Add 200 μ L of NaOH/SDS* - " BUFFER 2 " - Alkaline-SDS lysis must be done gently to avoid contamination of pDNA with chromosomal DNA - Cap the tube and mix by inversion 6X (do not vortex) - You should observe a clearing (lysis) of the cells * 0.2 N NaOH, 1% (w/v) SDS - The detergent sodium dodecyl sulfate (SDS) breaks the membrane's phosplolipid bilayer and dissolves proteins - The strong base sodium hydroxide (NaOH) denatures the proteins involved in maintaining the structure of the cell membrane and denatures DNA 6) Incubate tube on ice for 5 min… and not 1 sec more!!!
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