Isolation of Plasmid: Minipreparation

Short Protocol

Several Plasmid Miniprepation methods have been established such as the simple and quick boiling miniprep or those that use diatomaceous earth or cellulose. A boiling miniprep is sufficient if the plasmid will be used only for a restriction digestion. For sequencing, the DNA has to be very clean and it is recommended to use one of the commercial available miniprep kits such as the Quiagen miniprep kit or the BioRad miniprep kit (manual).

Equipment

  • heatblock/waterbath 37°C
  • microcentrifuges
  • pipetors 10, 100, 200, 1000
  • 1000, 200, 10 µl pipette tips
  • 1.5 ml sterile microcentrifuge tubes
  • 2.0 ml sterile microcentrifuge tubes
  • spin filter (Quantum Prep Plasmid Miniprep Kit, BioRad #732-6100)

Prepared solutions (Quantum Prep Plasmid Miniprep Kit, BioRad #732-6100)

  • cell resuspension solution
  • cell lysis solution
  • neutralization solution
  • wash buffer
  • sterile water (preheated to 37°C)

    To preserve the culture for future use, remove 200 µl bacterial culture and mix well with 200 µl sterile glycerol. Store at -70°C. Then continue with the plasmid prep. All centrifugation steps are carried out at max. speed.

    I. Wash culture:

    1. transfer 1.5 ml culture to a 1. 5ml sterile microcentrifuge tube
    2. spin for 30 sec
    3. remove supernatant medium
    4. add 200 µl cell resuspension solution to pellet
    5. resuspend pellet by vortexing or pipetting

    II. Lyse cells:

    1. add 250 µl lysis solution to resuspended cells
    2. cap tube and mix by inverting (10 times); DON'T let cells sit for longer than 5 min in lysis solution before you continue; solution should become clear
    3. add 250 µl neutralization solution
    4. cap tube and mix by inverting (10 times)
    5. incubate for 10 min; DON'T let cells sit for longer than 10 min in solution before you continue, precipitate should be formed

    III. Isolate plasmid:

    1. spin lysate at max. speed for 5 min in table top centrifuge
    2. place a spin filter into a clean 2.0 ml centrifuge tube
    3. transfer supernatant (which contains DNA) to spin filter; avoid debris
    4. add 200 µl of mixed matrix to spin filter and pipet up and down to mix
    5. spin for 30 sec
    6. discard the filtrate, add 500 µl wash buffer and spin for 30 sec
    7. repeat step III.6
    8. discard filtrate and spin for 2 min; it is very important that the matrix does not contain any war buffer
    9. remove spin filter and place in a sterile 1.5 ml centrifuge tube
    10. add 100 µl sterile water (preheated to 37°C); the water should be added so to the highest portion of the matrix
    11. incubate for 1 min
    12. spin for 1 min at top speed
    13. remove spin filter

    IV. Analysis:

    measure the OD at 260 and 280 nm and calculate the concentration of the plasmid DNA stock solution.