Corum:Miniprep

• SC 15:38, 25 June 2012 (EDT):

Overview

Gel Purification using Sigmas's GeneElute Plasmid Miniprep Kit.

Materials

• Miniculture
• 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

Procedure

1. Harvest the miniculture cells in a 2 ml tube by centrifugation. Remove all media by pipetting.
2. Resuspend pellet in 200 μL resuspension buffer by pipetting and vortexing.
3. Add 200 μL lysis solution. Gently invert 8 times. Incubate RT 4 mins.
4. Add 350 μL stop solution. Gently invert 8 times. Wait a moment, and invert another 8 times.
5. Spin 24,000g 3 min.
6. In the meantime, prepare the column system. Add 500 μL column preparation solution and spin maximum speed 1 min in a tabletop centrifuge.
7. 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.
8. Apply 500 μL optional wash solution to the column. Spin 30 s maximum speed. Discard the flowthrough.
9. Apply 500 μL wash solution to the column. Spin 30 s maximum speed. Discard the flowthrough.
10. Spin 1.5 min maximum speed to dry. Remove column and place in a new 1.5 ml tube.
11. Apply 50-100 μL water or buffer EB to the column (make sure not to touch the membrane). Incubate RT 10 min.
12. Elute DNA by spinning 2 min maximum speed.
13. Optional: combine elutions of the same plasmid DNA.
14. Optional: vacuum centrifuge at 60 °C$^\circ$C to evaporate liquid down to desired volume (usually 50-100 μL).

Notes

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References

Relevant papers, books, and websites

1. Invitrogen Life Technologies

Contact

• Sean P Corum

or instead, discuss this protocol.