IGEM:IMPERIAL/2006/Protocols/mini prep of culture

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Miniprep of cultures

  • Transfer 1.5ml of culture into eppendorf tubes.
  • Spin for 20seconds and remove supernatant.
  • Take out Solution I and III from the fridge with syringes. Solution II (if it is cold it precipitates) is on the bench. (use yellow pipette for syringes)
  • Add 100ul of solution I and resuspend the pellet by vortexing.
  • Add 200ul of solution II.
  • Add 150ul of solution III.
  • Mix them well by inverting the tubes. White precipitates appears.
  • Spin for 3min. In the meantime, label tubes.
  • Pour the supernatants in to the new labelled tubes, add 1ml of ethanol and mix.
  • Spin for another 3min and remove supernatant.
  • Spin to remove residual liquid and then resuspend in 50ul of MiliQ (1 sec. vortex. Don’t resuspend the pellet).
  • NB: If the culture was grown during the day (8hrs), then resuspend in 25μL to obtain approximately the same concentration.
  • Spin for 1min.
  • Ready to digest.

Solution 1

  • 90g glucose
  • 10ml 0.5M EDTA
  • 6.25ml 2M Tris pH8
  • make upto 500ml

Solution 2

  • For 500 mls
  • 50ml 10%SDS
  • 20ml NaOH

Solution 3

  • 147g potassium acetate
  • 57.5ml glacial acetic acid
  • make upto 500ml
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