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Unformatted text preview: Purification of Genomic DNA (gDNA) LABORATORY EXERCISE #4 Rev: 3/10/10 Purification of gDNA from Staphylococcal Food-Poisoning Isolates Principle(s) of the Procedure- The purification of DNA is an important achievement when one considers the fact that DNA represents only 3% of the dry weight of a bacterium (e.g. Escherichia coli ). - The remaining 97% consists of protein (50%), RNA (15-30%), polysaccharides (5-10%), lipids (10%), and numerous smaller organic metabolites. - The trick then is to use DNA's special responses to certain chemical treatments to decrease the initial contamination to a final purity of perhaps only 1-2% contamination. How well the DNA survives these treatments is a subject of considerable concern. Certain alterations occur during careless purification. In particular, DNA readily degrades upon exposure to excess heat, shear forces, and certain enzymes. Enzymatic degradation- Bacteria and even one's fingertips contain deoxyribonuclease (DNase), enzymes which specifically cleave the phosphodiester bonds between the nucleotides of the DNA chain. It is therefore necessary to prevent the action of these degradative enzymes at each step of the purification process. This can be accomplished in several ways: a. DNase activity depends on the presence of Mg ++ , which is a necessary cofactor for the action of the enzyme. Chelating agents , like ethylenediaminetetra-acetic acid ( EDTA ), tightly bind divalent cations and will inhibit this activity. b. DNases are inactivated at temperatures above 60 o C . Luckily, DNA doesnt melt until 85 - 90 o C, this makes it possible to inactivate DNAases without melting the DNA itself. c. Detergents such as sodium dodecyl sulfate ( SDS ) denature proteins (and hence DNase) without causing the DNA strands to separate. d. Proteinase K hydrolyzes proteins of the outer membrane, periplasmic space, cell membrane, and cytoplasm e. The organic extraction procedures (phenol and chloroform) remove proteins , and hence DNases, from the DNA. f. DNase activity is reduced by incuation of samples at 0-4 o C [keeping samples on ice.] 1 Step 1 - Cell Lysis 1) Inoculation and Growth of Broth Cultures- Inoculate 15 mL of LB broth with isolate- Incubate for 18 hr at 37C in shaker incubator at 100 rpm 2) Harvest the Cells- Sediment the cells by centrifugation at 10,000 x g for 15 min- Pour off and discard the supernatant- Remove the remaining fluid using a pipette gun, being careful not to disturb the pellet (the small off-white smear located at the tubes side/bottom beneath the hinge) 3) Suspension of the Cells in SMTB buffer w/ Lysozyme and EDTA- Add 500-1000 L SMTB buffer to the cell pellet- COMPLETELTY resuspend the cell pellet thoroughly by pipetting up and down several times with a micropipette gun, until the suspension is homogeneous (no clumps) 4) Enzymatic Digestion of the Cell Wall by Lysozyme- Add 10 L Lysozyme [10 g/mL]- EDTA contained in SMTG aids lysozyme in degradation of cell wall...
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