Team:CSU Fort Collins/Notebook/Protocols=Miniprep

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Revision as of 17:05, 17 October 2014

Plasmid Miniprep

Plasmid Miniprep Protocol

Show Table of Contents

Before starting:

  • Add RNase A to Resuspension Buffer (R3) according to the instructions on the label; mix well
  • Mark on the label that RNase A is added. Store Buffer R3 with RNase A at 4 °C
  • Warm Lysis Buffer (L7) briefly at 37 °C to redissolve any particulate matter
  • Add 100% ethanol to Wash Buffer (W9) and Wash Buffer (W10) according to the instructions on each label; mix well
  • Store wash buffers with ethanol at room temperature
  1. Harvest
    • Sediment the cells by centrifuging 1 - 5 mL of the overnight LB culture (use 1-2 x 109 E.coli cells for each sample)
    • Remove all medium
  2. Resuspend
    • Add 250 μL Resuspension Buffer (R3) with RNase A to the cell pellet
    • Resuspend pellet until it is homogeneous
  3. Lyse
  4. Note: This step is time sensitive.
    • Add 250 μL Lysis Buffer (L7)
    • Mix gently by inverting the capped tube five times (Do NOT vortex)
    • Incubate the tube at room temperature for 5 minutes
  5. Precipitate
    • Add 350 μL Precipitation Buffer (N4)
    • Mix immediately by inverting the tube, or for large pellets, vigorously shaking the tube, until the mixture is homogeneous (Do NOT vortex)
    • Centrifuge the lysate at >12,000 X g for 10 minutes
  6. Bind
    • Load the supernatant from step 4 onto a spin column in a 2-mL wash tube
    • Centrifuge the column at 12,000 X g for 1 minute
    • Discard the flow-through and place the column back in the wash tube
  7. Wash and Ethanol Removal
    • Add 700 μL Wash Buffer (W9) with ethanol to the column
    • Centrifuge the column at 12,000 X g for 1 minute
    • Discard the flow-through and place the column back into the wash tube
    • Centrifuge the column at 12,000 X g for 1 minute
    • Discard the was htube with the flow-through
  8. Elute
    • Place the spin column in a clean 1.5-mL recovery tube
    • Add 75 μL of preheated TE Buffer (TE) to the center of the column
    • Incubate the column for 1 minute at room temperature
  9. Recover
    • Centrifuge the column at 12,000 X g for 2 minutes
    • The recovery tube now contains the purified plasmid DNA
    • Discard the column
    • Store plasmid DNA at 4 °C (short-term) or store the DNA in aliquots at -20 °C (long-term)

Thank you to Life Technologies for this protocol.



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