CAVEAT!!! Usually we do these procedures with a pure culture ◦ Grow bacteria in broth ◦ Streak on plate ◦ Grow single colony on broth

Microbial DNA Extraction Bridges 2014

Cheek Cell

Bacterial Cell

Step One: Lysis Breaking apart the cell Physical ◦ Vortex ◦ MicroBeads Chemical ◦ MD1  SDS  Disrupts membrane and denatures proteins

Step Two: Separation from Debris Centrifugation Precipitation of Proteins More Centrifuging Supernatant Pellet

Step Three: Clean DNA Bind to spin filter ◦ Salt solution ◦ Silica filter Wash with ethanol ◦ Inhibits PCR Elute off of filter

Contamination It is important when working with bacteria to use sterile technique ◦ Wipe work area, pipets, and pipet boxes with EtOH ◦ Use aerosol filter pipets ◦ Keep tips covered ◦ Change tips  Especially if potentially contaminated ◦ Change gloves often! ◦ Keep tubes closed as much as possible ◦ Do NOT touch inside of tubes STAY ORGANIZED!