1. 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 hours after initiation

2. 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

3. 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

4. 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