Zymo miniprep classic

Zymo miniprep classic kdorfman Thu, 09/15/2022 - 19:53

For many labs

Before beginning, add 95% ethanol to Plasmid Wash Buffer 4:1 Also, once RNAse has been added to P3 (yellow), keep it at 4C.

Protocol

  • Spin 0.5 - 5 mL bacterial culture in 1.5 mL tube 20 sec full speed

  • Add 200 uL P1 (red), resuspend pellet

  • Add 200 uL P2 (green); mix by inverting 2-4 times. Clear, viscous, purple solution indicates cell lysis

  • Add 350 uL P3 (yellow). Mix thoroughly. DO NOT VORTEX. Turns yellow when neutralization is complete.

  • Incubate RT 1-2 min

  • Spin 2 min

  • Put column into collection tube, transfer supernatant. DO NOT DISTURB GREEN PELLET (I found it wasn't green, but OK)

  • Spin 30 sec

  • Discard flow through, put column back into emptied collection tube

  • Add 200 uL Endo-Wash Buffer to the column, spin 30 sec

  • Add 400 uL Plasmid wash buffer to the column, centrifuge 1 min

  • Transfer column to clean 1.5 mL microcentrifuge tube

  • Add 30 uL DNA Elution Buffer

  • Spin 30 seconds. Plasmid DNA is in the collection tube

Students need per reaction1

  • 1 mL (?) overnight culture of transformed cells

  • 1 column, 1 2-mL collection tube

  • sterile 1.5 mL tube

  • ~200 uL P1 (red)

  • ~200 uL P2 (green)

  • ~350 uL P3 (yellow)

  • ~200 uL Endo-Wash-Buffer

  • ~400 Plasmid Wash Buffer

  • ~30 uL DNA Elution Buffer


  1. All the ingredients are sold separately, so it's OK to give students some extra to allow for pipetting errors. ↩︎