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Plasmid MiniPrep - Qiagen

Created by Ravishankar Madhu

Last updated: 2023-07-10

Description

This protocol describes how to complete a MiniPrep in which plasmid DNA is extracted 2mL of Bacterial Culture using the Qiagen MiniPrep Kit.

Reagents and Materials

  • Bacterial Culture
  • QIAprep Spin Miniprep Kit
    • P1 Buffer
    • P2 Buffer
    • N3 Buffer
    • PB Buffer
    • PE Buffer
    • EB Buffer
    • QIAprep spin column
  • 2.0mL Eppendorf Tubes
  • 1.5mL Eppendorf Tubes

Things to Note before starting

Handling the Kit

You should add LyseBlue reagent to the P1 Buffer at a ratio of 1:1000. You should also add the provided RNase A solution to the P1 Buffer. After the P1 Buffer has been supplemented with those two reagents, it should be stored in the 4C Fridge. The PE Buffer has to be supplemented with 100% ETOH. Follow the instructions provided with the kit and label the bottles appropriately before using either buffer.

Amount of Cells

This protocol presumes you will use 2mL of Bacterial Culture as an input. I would not recommend using more than 2mL.

Estimated Timing

This protocol takes 30-90 minutes to complete and is best done with 8-16 samples but can be done with up to 24 samples.

Procedure

  1. Aliquot up to 2mL of Bacterial Culture into 2.0mL Eppendorf Tubes.
  2. Pellet the bacterial culture by spinning down at 13,000 rcf for 10 minutes at 4C.
  3. Aspirate off the bacterial culture without disrupting the cell pellet.
  4. Resuspend the bacterial pellet in 250uL of P1 Buffer.
  5. Add 250uL of P2 Buffer.
  6. Invert the tube 4 times. The solution should turn blue. 1
  7. Let the solution incubate for 4 minutes at room temperature.
  8. Add 350uL of N3 Buffer.
  9. Mix by inverting 4 times. The solution should turn milky white as a precipitate should form.
  10. Spin at 13,000 rcf for 10 minutes.
  11. Transfer the supernatant into a QIAprep Spin column. Try to avoid disrupting the pellet at the bottom of the tube. 2
  12. Spin the column for 1 minute at 13,000 rcf. Discard the flow through. 3
  13. Add 500uL of PB Buffer to the column.
  14. Spin the column for 1 minute at 13,000 rcf. Discard the flow through.
  15. Add 750uL PE Buffer to the column.
  16. Spin the column for 1 minute at 13,000 rcf. Discard the flow through.
  17. Spin the empty column for 3 minutes at 13,000 rcf. Discard any flow through.
  18. Transfer the column into a clean 1.5mL Eppendorf Tube.
  19. Add 50uL of EB buffer to the column.
  20. Let columns sit at room temperature for 2 minutes.
  21. Spin samples for 1 minute at 13,000 rcf. Discard the column.

Next Steps

The plasmid DNA can be quantified using the NanoDrop. The plasmid DNA can be stored in a -20C Freezer until further use in a Virus Prep

Appendix

Footnotes

  1. The solution will only turn blue if the P1 Buffer was supplemented with the LyseBlue Reagent. ↩

  2. You are transferring the SUPERNATANT not the Pellet. The DNA is located in the Supernatant. ↩

  3. The flow through can be stored in a plastic bottle along with the flow through from other DNA extractions. Once the bottle is full, it can be tossed down the sink. ↩