- SC 15:38, 25 June 2012 (EDT):
Miniprep plasmid DNA isolation using Sigmas's GeneElute Plasmid Miniprep Kit.
- 1-5 mL overnight miniculture cells (E. coli)
- GeneElute miniprep column
- Resuspension buffer
- Lysis solution
- Stop solution
- Column preparation solution
- Wash solution
- Optional wash solution
- Elution buffer or H2O
- Harvest the miniculture cells in a 2 ml tube by centrifugation. Remove all media by pipetting.
- Resuspend pellet in 200 μL resuspension buffer by pipetting and vortexing.
- Add 200 μL lysis solution. Gently invert 4-6 times. Incubate RT 4 mins.
- Add 350 μL stop solution. Gently invert 4-6 times. Wait a moment, and invert another 4-6 times.
- Spin 24,000g 3 min.
- In the meantime, prepare the column system. Add 500 μL column preparation solution and spin maximum speed 1 min in a tabletop centrifuge.
- Leaving the cellular debris behind, remove the supernatant by decanting or pipetting and apply to the column membrane. Spin 30 s maximum speed. Discard the flowthrough.
- Apply 500 μL optional wash solution to the column. Spin 30 s maximum speed. Discard the flowthrough.
- Apply 500 μL wash solution to the column. Spin 30 s maximum speed. Discard the flowthrough.
- Spin 1.5 min maximum speed to dry. Remove column and place in a new 1.5 ml tube.
- Apply 50-100 μL water or buffer EB to the column (make sure not to touch the membrane). Incubate RT 10 min.
- Elute DNA by spinning 2 min maximum speed.
- Optional: combine elutions of the same plasmid DNA.
- Optional: vacuum centrifuge at 60 °C to evaporate liquid down to desired volume (usually 50-100 μL).
Please feel free to post comments, questions, or improvements to this protocol. Happy to have your input!
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