Toronto/Lab Protocols/Overnight

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== Miniprep ==
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== Overnight (O/N) Preparation: ==
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Small Scale Plasmid Preparation (1 – 2 hours)
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#Put 2.5 mL of LB Broth into a 15 mL tube. (Make sure to flame the broth)
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#Scrape off a single colony from a plate with a pipette tip and eject tip into the tube.
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'''Overnight (O/N) Preparation:'''
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#Add 2.5 μL of the appropriate antibiotics. (Check spreadsheet)
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#Incubate overnight.
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#1. Put 2.5 mL of LB Broth into a 15 mL tube. (Make sure to flame the broth)
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#2. Scrape off a single colony from a plate with a pipette tip and eject tip into the tube.
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#3. Add 2.5 μL of the appropriate antibiotics. (Check spreadsheet)
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#4. Incubate overnight.
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'''Miniprep Procedure:'''
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#1. Put 1.5 mL O/N into a 1.5 mL Eppendorf tube.
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#2. Centrifuge for 1 minute.
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#3. Aspirate the supernatant.
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#4. In order to increase the concentration of DNA, add the rest of the o/n into the same tube and centrifuge for 1 minute again.
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#5. Aspirate the supernatant and resuspend cells with 250 μL Resuspension Solution (red). Vortex until pellet dissolves.
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#6. Add 250 μL Lysis Solution and mix with pipette. (shake with hand)
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#7. Sit for 3-5 minutes.
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#8. Add 350 μL Neutralization Solution (blue) and invert tube with hand 4-6 times.
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#9. Centrifuge for 5 minutes.
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#10. Pour supernatant in a column tube.
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#11. Centrifuge for 1 minute and throw away supernatant.
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#12. Add 500 μL Wash Solution.
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#13. Centrifuge for 1 minute and throw away supernatant.
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#14. Add 500 μL Wash Solution.
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#15. Centrifuge for 1 minute and throw away supernatant.
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#16. Put blue column in a fresh centrifuge tube. (make sure to label)
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#17. Add 50 μL Elution Buffer.
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#18. Incubate for 2 minutes at room temperature, then centrifuge for 2 minutes.
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#19. Throw away blue column.
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#20. Place plasmid into the freezer.
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Jump to [http://igem.skule.ca/lab/protocols/miniprep.htm BlueGenes]
Jump to [http://igem.skule.ca/lab/protocols/miniprep.htm BlueGenes]

Latest revision as of 03:38, 27 October 2007

Overnight (O/N) Preparation:

  1. Put 2.5 mL of LB Broth into a 15 mL tube. (Make sure to flame the broth)
  2. Scrape off a single colony from a plate with a pipette tip and eject tip into the tube.
  3. Add 2.5 μL of the appropriate antibiotics. (Check spreadsheet)
  4. Incubate overnight.

Jump to BlueGenes

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