Video: Plasmid Maxiprep Procedure

The GenElute™ HP Plasmid Maxiprep kit offers a simple, rapid, and cost-effective method for isolating plasmid DNA from recombinant E. coli cutures. The recovered plasmid DNA is ready for immediate use in downstream applications, such as restriction digestion, ligation, sequencing, PCR, transformation, and transfection.

  • Culture Volume - 150 mL bacterial culture
  • Yield - Up to 1.2 mg yield of high-copy plasmid DNA
  • Speed - From harvested bacterial culture to pure plasmid DNA in 30 minutes or less
  • Flexible - Offers the flexibility of a vacuum or spin format, does not rely on gravity flow or ultracentrifugation
  • Economic - Lower cost per prep plus higher yields per prep equals double savings

Plasmid DNA Maxiprep Steps

  1. Pellet 150 mL of an overnight culture by centrifugation.

  2. Add 12 mL of resuspension solution.

  3. Pipette up and down or vortex to completely resuspend the bacterial pellet.

  4. Add 12 mL of the lysis buffer.

  5. Let mixture sit for 5 minutes.

  6. Gently invert 6-8 times. Do not vortex.

  7. Prepare the filter syringe.

  8. Add 12 mL of the neutralization solution to the mixture.

  9. Gently invert 4-6 times.

  10. Add 9 mL of binding solution to the mixture.

  11. Invert 1-2 times.

  12. Pour the mixture into the barrel of the filter syringe.

  13. Allow the lysate to sit for 5 minutes.

  14. Gently invert 6-8 times. Do not vortex.

  15. Prepare the filter syringe.

  16. Add 12 mL of the neutralization solution to the mixture.

  17. Gently invert 4-6 times.

  18. Add 9 mL of binding solution to the mixture.

  19. Invert 1-2 times.

  20. Pour the mixture into the barrel of the filter syringe.

  21. Allow the lysate to sit for 5 minutes.

  22. Add 12 mL of the column preparation solution to the binding column, allowing it to pass through.

  23. Following the 5-minute incubation, expel the clear lysate into a fresh collection tube. It is not necessary to force residual lysate through the filter syringe.

  24. Pour the lysate into the binding column and allow it to pass through.

  25. Add 12 mL of wash solution 1 to the binding column and allow it to pass through.

  26. Add 12 mL of wash solution 2 to the binding column and allow it to pass through.

  27. Leave the vacuum on for 10 minutes to dry the column. If more than 6 maxiprep columns, dry for at least 20 minutes.

  28. Transfer the binding column to a clean collection tube.

  29. Add 3 mL of elution solution to the column and centrifuge for 5 minutes.

  30. The plasmid DNA is now present in the eluate.

Materials
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