Team:Queens Canada/mini

MiniPrep (Collin) · Benchling

MiniPrep (Collin)

Introduction

Protocol is designed for purification of up to 20 µg of high-copy plasmid DNA from 1-5 mL overnight cultures of E. coli in LB medium.

Materials

    • MiniPrep Kit
    • Sample
    • Centrifuge
    • Eppendorf tubes
    • Pipette and tips

Procedure

  1. Grab 2X 1.5mL Eppendorf tubes per overnight culture
  1. Label tubes
  1. Pipette 1 mL into 1.5 mL Eppendorf tubes
  1. Centrifuge 1 min @ 16 g
  1. Pour out supernatant into empty flask.
  1. Repeat until conical tube is empty.
  1. With more precise pipette (smaller), extract the remaining liquid without extracting the pellet.
  1. Add 250 uL of resuspension solution and mix until dissolved.
  1. Add 250 uL of lysis solution to each, then invert 6X
  1. Open caps to see if the solution is slimy - that's good!
  1. Add 250 uL of neutralization buffer to each and then immediately invert.
  1. Centrifuge for 5 min @ 16 g.
  1. Label spin columns
  1. Transfer supernatant NOT white chromosomal DNA into tubes
  1. Spin for 1 min @ 16 g
  1. Add 500 mL of wash solution
  1. Spin for 1 min.
  1. Repeat steps 15, 16.
  1. Then centrifuge empty.
  1. Then elute.
  1. Grab new 1.5 eppendorf tubes, spin columns into tubes.
  1. Pipette 50 uL ddH2O direct onto filter and let sit for 2 min.
  1. Centrifuge for 1 min.
  1. Now pipette the 50 uL tha passes through the filter into the next tube.
  1. Wait 2 minutes.
  1. Repeat steps 24-26 until all filters have been used. (concentrates plasmids)
  1. Resulting plasmid solution in 1.5 mL epp tubes. All clean up goes in biohazard bin.
  1. NANODROP