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Before you start, remove the pipette tip inside the overnight tubes using a pipette.

  1. Add 1.5mL of overnight culture to microcentrifuge tube. Pipet overnight culture up and down to mix before adding. Pellet the cells by centrifuging for 1-2 minutes at maximum speed. Decant the supernatant, then add another 1.5mL of overnight culture and repeat centrifugation. Decant the supernatant and remove all medium residue by pipet.
    • From the extra overnight culture, make glycerol stocks (see Lab Protocol Glycerol Stocks)
  2. Completely resuspend the cell pellet in 200μl of MX1 buffer (found in the cold room) by pipeting up and down until there are no visible clumps.
  3. Add 250μl MX2 buffer and gently invert 4-6 times to lyse the cells. Incubate at RT for 1-5 minutes. (Do not vortex)
  4. Add 350μl MX3 buffer to neutralize lysate and gently mix solution. Immediately centrifuge for 10 minutes at maximum speed. MIX WELL. LOTS. GENTLY.(Be sure to mix the two layers with pipet prior to centrifuging)
  5. Transfer supernatant to a spin column. Centrifuge for 30-60 seconds at 5000RPM. Discard flow-through.
  6. Wash column with 500μl WN buffer by centrifuging for 30-60 seconds at 9000RPM. Discard flow-through.
  7. Wash column with 700μl WS buffer by centrifuging for 30 seconds at 9000RPM. Discard flow-through.
  8. Centrifuge the column at 13000RPM for another two minutes to remove residual ethanol.
  9. Place column into a new labeled microcentrifuge tube. Add 50μl of nuclease free water. Be sure water is suspended onto center of the membrane and is completely absorbed.
  10. Let column stand at RT for 10 minutes to increase recovery yield. Centrifuge for 30 seconds at 13000RPM to elute.
  11. Nanodrop 1μl of DNA and write DNA concentration to microcentrifuge tube before storing. Also record the purity concentration which should be in the range of 1.8-2.0. (Be sure to blank Nanodrop before loading DNA)