Plasmid Purification Miniprep Process of growing selected bacteria in a liquid culture 1) purifies the plasmids out of the bacteria 2) amplifies the plasmids for further experiments Liquid culture must be warm, contain nutrients, & be oxygenated. Harvesting must be done in the growth phase 16-24 hours after initiation

Plasmid Purification Bacteria is moved from LB broth to a minicentrifuge tube and spun Moves bacteria to the bottom Supernatant removed and replaced with a alkaline lysis buffer Denatures the plasma membrane and releases DNA pH is neutralized with a moderate acid Allows reannealing of any separated DNA Centrifugation is used to move heavier debris and gDNA to the bottom of the tube Purification to remove salts Alcohol precipitation or column chromotagraphy

DNA Quantification Gel Quantification Intensity (darkness) of the band produced is indicative to the amount of DNA Can use a molecular mass ruler Difference in distance traveled can be due to nicked plasmids, open plasmids, or supercoiled plasmids

Spectrophotometric Quantification Spectrophotometers can be used to quantify based on light absorbance DNA absorbs light at a frequency of 1 at 260nm (A260) within the UV range A 50mg/ml solution with an absorbance of 0.5mg has an overall DNA concentration of 25mg/ml gDNA and RNA absorb at different frequencies A protein contaminant that absorbs at 280nm is introduced to aid in confirmation of plasmid DNA