Toronto/Lab Protocols/Overnight

From 2007.igem.org

(Difference between revisions)
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'''Overnight (O/N) Preparation:'''
'''Overnight (O/N) Preparation:'''
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#1. Put 2.5 mL of LB Broth into a 15 mL tube. (Make sure to flame the broth)
+
#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.
+
#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)
+
#Add 2.5 μL of the appropriate antibiotics. (Check spreadsheet)
-
#4. Incubate overnight.
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#Incubate overnight.
'''Miniprep Procedure:'''
'''Miniprep Procedure:'''
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#1. Put 1.5 mL O/N into a 1.5 mL Eppendorf tube.
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#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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#Centrifuge for 1 minute.
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#3. Aspirate the supernatant.
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#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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#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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#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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#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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#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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#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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#Centrifuge for 5 minutes.
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#10. Pour supernatant in a column tube.
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#Pour supernatant in a column tube.
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#11. Centrifuge for 1 minute and throw away supernatant.
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#Centrifuge for 1 minute and throw away supernatant.
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#12. Add 500 μL Wash Solution.
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#Add 500 μL Wash Solution.
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#13. Centrifuge for 1 minute and throw away supernatant.
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#Centrifuge for 1 minute and throw away supernatant.
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#14. Add 500 μL Wash Solution.
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#Add 500 μL Wash Solution.
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#15. Centrifuge for 1 minute and throw away supernatant.
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#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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#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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#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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#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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#Throw away blue column.
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#20. Place plasmid into the freezer.
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#Place plasmid into the freezer.
Jump to [http://igem.skule.ca/lab/protocols/miniprep.htm BlueGenes]
Jump to [http://igem.skule.ca/lab/protocols/miniprep.htm BlueGenes]

Revision as of 03:37, 27 October 2007

Miniprep

Small Scale Plasmid Preparation (1 – 2 hours)

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.

Miniprep Procedure:

  1. Put 1.5 mL O/N into a 1.5 mL Eppendorf tube.
  2. Centrifuge for 1 minute.
  3. Aspirate the supernatant.
  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.
  5. Aspirate the supernatant and resuspend cells with 250 μL Resuspension Solution (red). Vortex until pellet dissolves.
  6. Add 250 μL Lysis Solution and mix with pipette. (shake with hand)
  7. Sit for 3-5 minutes.
  8. Add 350 μL Neutralization Solution (blue) and invert tube with hand 4-6 times.
  9. Centrifuge for 5 minutes.
  10. Pour supernatant in a column tube.
  11. Centrifuge for 1 minute and throw away supernatant.
  12. Add 500 μL Wash Solution.
  13. Centrifuge for 1 minute and throw away supernatant.
  14. Add 500 μL Wash Solution.
  15. Centrifuge for 1 minute and throw away supernatant.
  16. Put blue column in a fresh centrifuge tube. (make sure to label)
  17. Add 50 μL Elution Buffer.
  18. Incubate for 2 minutes at room temperature, then centrifuge for 2 minutes.
  19. Throw away blue column.
  20. Place plasmid into the freezer.

Jump to BlueGenes

Retrieved from "http://2007.igem.org/wiki/index.php/Toronto/Lab_Protocols/Overnight"