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Genomic DNA - Anatomy Notes
Anatomy And Physiology I/Lab (BIOL 3320)
Nova Southeastern University
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Goal - purify genomic DNA from bacteria ○ Total DNA - plasmids + chromosomes Used for PCR but cannot tell if any of the product came from the chromosome Used to determine if Bacillus strains have an amyE gene (and other genes) with significant homology to the Bacillus licheniformis primers Followed by sequencing amyE gene, synthesizing amyE gene, and cloning amyE gene
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Process ○ Lyse the cells and the nuclei. The cellular proteins are then removed by a salt precipitation step, which precipitates the proteins but leaves the high molecular weight genomic DNA in solution
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○ Genomic DNA is concentrated and desalted by isopropanol precipitation
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Lysozyme - degrades peptidoglycan cell wall of gram positive bacteria ○ Lysostaphin and mutanolysin - cell lysis enzymes required for some bacteria
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- Goal is to pellet out the protein and have the supernatant contain the genomic DNA DNA is insoluble in ethanol and isopropanol Isopropanol - allows precipitation of larger species and lower concentrations of nucleic acids than ethanol; salts tend to co-precipitate with DNA because salt is less soluble than in ehanol
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○ Followed up by 70% ethanol wash to desalt
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Procedure ○ Pellet cells via centrifugation of the bacterial culture (2x) ○ Wash with TE ○ Centrifuge If gram positive bacterium, resuspend in EDTA TRIS EDTA EDTA inhibits DNAses by chelating Mg2+ and other metal ions required for nucleases
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□ If gram negative bacterium, add Nuclei Lysis Solution
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○ Add lysozyme
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Genomic DNA Prep
Thursday, October 27, 2022 8:12 PM
Genomic DNA - Anatomy Notes
Course: Anatomy And Physiology I/Lab (BIOL 3320)
University: Nova Southeastern University
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