Getting Started

Before proceeding: inoculate liquid media and grow sample using the Culturing E. coli protocol.

 

 

Materials

Consumables:

  • Binding buffer
  • II-P spin column
  • Microcentrifuge tubes
  • Ice
  • Zymo Research Mini-Prep Kit

Hardware:

  • Centrifuge

Procedure

  1.  Set up all the tubes necessary for the mini-prep in a tube rack and number all the tubes (i.e. label all the tubes in the first column of the tube rack ‘1’ in order to track the flow of the procedure).
    • 3 microcentrifuge tubes
    • 1 spin column
  2.  Centrifuge 3-5 ml of bacterial culture in total
  3.  In a clear 1.5 ml tube, spin down 1 ml of culture at a time at full speed for 15-20 seconds. Aspirate off supernatant.
  4.  Add 200 μl of ZymoPURE™ P1 (Red) to the bacterial cell pellet and resuspend completely by vortexing or pipetting.
  5.  Add 200 μl of ZymoPURE™ P2 (Green) and immediately mis by gently inverting the tube 2-4 times. Do not vortex! Let sit at room temperature for 1-2 minutes. Cells are completely lysed when the solution appears clear, purple, and viscous.
  6.  Add 400 μl of ZymoPURE™ P3 (Yellow) and mix thoroughly by inversion. Do not vortex! Invert the tube an additional 3-4 times after the sample turns completely yellow. Incubate at room temperature for 1-2 minutes. The sample will turn yellow when the neutralization is complete and a yellowish precipitate will form.
  7.  Centrifuge the neutralized lysate for 2-4 minutes at +11,000 x g.
  8.  Place a Zymo-Spin IIN column into a collection tube and transfer supernatant. Be careful not to disturb the yellow pellet and avoid transferring any cellular debris to the new tube.
  9.  Centrifuge the Zymo-Spin IIN assembly for 30 seconds at +11,000 x g.
  10.  Discard the flow through and return Zymo-Spin IIN column to the same collection tube.
  11.  Add 200 μl of Endo-Wash Buffer to the column and centrifuge for 30 seconds at +11,000 x g.
  12.  Add 400 μl of Plasmid Wash Buffer to the column and centrifuge for 1 min at +11,000 x g.
  13.  Transfer Column into a clean 1.5 microcentrifuge tube and add 30 μl of DNA Elution Buffer directly to the column matrix and incubate for 10 minutes at room temperature.
  14.  Centrifuge for 30 seconds at +11,000 x g to elute plasmid DNA.