Before proceeding: inoculate liquid media and grow sample using the Culturing E. coli protocol.
- Binding buffer
- II-P spin column
- Microcentrifuge tubes
- Zymo Research Mini-Prep Kit
- 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
- Centrifuge 3-5 ml of bacterial culture in total
- 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.
- Add 200 μl of ZymoPURE™ P1 (Red) to the bacterial cell pellet and resuspend completely by vortexing or pipetting.
- 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.
- 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.
- Centrifuge the neutralized lysate for 2-4 minutes at +11,000 x g.
- 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.
- Centrifuge the Zymo-Spin IIN assembly for 30 seconds at +11,000 x g.
- Discard the flow through and return Zymo-Spin IIN column to the same collection tube.
- Add 200 μl of Endo-Wash Buffer to the column and centrifuge for 30 seconds at +11,000 x g.
- Add 400 μl of Plasmid Wash Buffer to the column and centrifuge for 1 min at +11,000 x g.
- 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.
- Centrifuge for 30 seconds at +11,000 x g to elute plasmid DNA.