Team:Washington/ISOLATION OF PLASMID

From 2013.igem.org


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 for 1 minute ( 17,000g = 13,000rpm)
    • Use enough culture to end up with 75 uls of pelleted cells
    • Start with 1.5 mL
    • Carefully pour off the supernatant without disturbing the pelleted cells
    • Then add antoher 1.5mL on top
  3. Resuspend the pelleted cells in 250 μL of refrigerated buffer P1.
  4. Add 250 μL of buffer P2 and thoroughly mix the tube by inverting 4-6 times.
  5. Add 350 μL of buffer N3 and immediately (but gently) mix the tube by inverting 4-6 times.
  6. Centrifuge the sample at ~17000 X g for 10 minutes. - Make sure to balance the centrifuge or risk damaging the brake on the centrifuge.
  7. Pour/Pipet the supernatant into a spin column (blue).
  8. Centrifuge the sample for ~ 1 minute. (Discard the flow-through.) - Repeat step 9 before discard.
  9. Wash the sample by adding 500 μL buffer PB and centrifuge for ~ 1 minute (optional).
  10. Wash the sample by adding 750 μLbuffer PE and centrifuge for ~ 1 minute.
  11. Discard the flow-through and centrifuge the sample for an additional 1 minute.
  12. To Elute the DNA, place the spin column in a clean 1.5 mL (labeled) microcentrifuge. Add 30 μL of buffer EB and let Stand for 1 minute!
    1. It is better to add 15 ul of EB buffer then wait 1-2 minutes, spin, add another 15 ul of EB buffer then wait 1-2 minutes and spin
    2. Waiting more than 15 minutes - 1 hours helps increase yield
  13. Centrifuge the sample for 1 minute.
  14. Record the DNA concentration using the nanodrop program (see Nanodrop Protocol).
  • Open the nanodrop program on the lab computer.
  • Select "DNA"
  • Once the program loads, pipet 1 ul of distilled H20 onto the machine.
  • Hit "OK" in order to run the sample to initialize. Wipe off the remaining water with a kimwipe.
  • Pipet 1 ul of EB buffer onto the machine. Hit "Measure" in order to run the sample. Wipe off the remaining buffer with a kimwipe.
  • Pipet 1 ul of a miniprep sample onto the machine. Hit "Measure" in order to run the sample. Wipe off the remaining sample with a kimwipe.
  • Record the concentration of the miniprep (ng/ul).
  • Repeat the process of pipeting a sample and "collected" until all samples have been measured.
  1. Label tube with Sample Name, Initials, Date and DNA concentration.

Equipment Necessary

  1. Centrifuge
  2. QIAGEN Miniprep Kit
  3. Microcentrifuge tubes

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