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CHROMOSOMAL & PLASMID DNA ISOLATION FROM BACTERIAL CELLS PLASMIDS Small extrachromosomal circular dsDNA molecules Not crucial for bacterial survival Confers bacteria w/ selective advantage » Synthesis of toxins - pathogenicity » Antibiotic resistance - Resistance-transfer factors RTFs » Synthesis of the F pilus » Metal tolerance » Novel enzymatic activities » Synthesis of Adhesins Utilization as cloning vehicles in biotechnology BACTERIAL PLASMIDS CHROMOSOMAL DNA ISOLATION Two Methods: 1. Spooling 2. Extraction Spooling DNA Extraction The basic steps of Chromosomal DNA isolation are: 1. Disruption of the cellular structure Separation of the soluble DNA from cell debris and other insoluble material and Purification of the DNA of interest from soluble proteins and other nucleic acids 2. 3. Culture centrifugation PLASMID DNA ISOLATION FROM BACTERIAL CELLS For the selective isolation of plasmid DNA, cells are exposed to NaOH and a strong detergent (alkaline lysis) to denature de Chromosomal DNA (i.e. the 2 strands are separated). An acidic solution of sodium acetate is then added to neutralize the solution. At this point, most of the cell membrane material and the genomic DNA are spilled out and precipitate . The selective precipitation of Genomic DNA is based in its higher molecular weight. DNA Isolation Kits Kits for plasmid DNA purification are commercially available and provide faster and more accurate isolation and purification procedures They are mostly based DNA-binding in columns after cell lysis and precipitation of insoluble materials Advantages: Faster and higher yields of DNA We are going to use a Commercial Kit for plasmid DNA isolation from E.coli pDSRed2, containing the red fluorescent gene All samples containing DNA should be kept on ice during the isolation process