Team:Washington/Protocols/Plas DNA.

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Revision as of 06:14, 29 September 2012


Isolation of Plasmid DNA (miniprep)

  1. Gently vortex overnight culture(s) to mix cells.
  2. Pipet ~1 mL of cells into a labeled microcentrifuge tube. Pellet the cells by centrifuging at 6000 X g.
  3. Carefully pour off the supernatant without disturbing the pelleted cells.
  4. Resuspend the pelleted cells in 250 μL of refrigerated buffer P1.
  5. Add 250 μL of buffer P2 and thoroughly mix the tube by inverting 4-6 times.
  6. Add 350 μL of buffer N3 and immediately (but gently) mix the tube by inverting 4-6 times.
  7. Centrifuge the sample at ~17000 X g for 10 minutes.
  8. Pour/Pipet the supernatant into a spin column (blue).
  9. Centrifuge the sample for ~ 1 minute. Discard the flow-through.
  10. Wash the sample by adding 500 μL buffer PB and centrifuge for ~ 1 minute.
  11. Wash the sample by adding 750 μLbuffer PE and centrifuge for ~ 1 minute.
  12. Discard the flow-through and centrifuge the sample for an additional 1 minute.
  13. To Elute the DNA, place the spin column in a clean 1.5 μL (labeled) microcentrifuge. Add 30 μL of buffer EB and let Stand for 1 minute!
  14. Centrifuge the sample for 1 minute.
  15. Record the DNA concentration using the nanodrop program.
    • Open the nanodrop program on the lab computer.
    • Select "DNA"
    • Once the program loads, pipet 1 ul of distilled H20 onto the machine.
    • Hit "collect" in order to run the sample to clean the machine.Wipe off the remaining water with a kimwipe.
    • Pipet 1 ul of EB buffer onto the machine. Hit "collect" in order to run the sample to clean the machine.Wipe off the remaining buffer with a kimwipe.
    • Pipet 1 ul of a miniprep sample onto the machine. Hit "collect" in order to run the sample to clean the machine.Wipe off the remaining sample with a kimwipe, and record the concentration of the miniprep (ng/ul).
    • Repeat the process of pipeting a sample and "collected" until all samples have been measured.
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