Team:Glendale CC AZ/igem.org

From 2013.igem.org

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==Alkaline Lysis Plasmid Miniprep==
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'''Purpose:''' To isolate plasmid DNA from transformed cells.
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'''Reagents:'''
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*Lysis solution (50mM glucose, 25mM TrisHCl pH 8, 10 mM EDTA)
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: 9.0mL dH2O
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: 0.25mL 1M TrisHCl pH 8
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: 0.25mL 0.5 M Na2EDTA
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: 0.50mL 20% glucose
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*SDS/NaOH (prepare fresh)
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: 880ul dH2O
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: 100ul 10% SDS
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: 20ul 10M NaOh
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*Acetate solution
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: 60mL 5M potassium acetate
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: 11.5mL glacial acetic acid
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: 28.5mL dH2O
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'''Materials:'''
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*1 colony of transformed cells
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*5mL LB/chloramphenicol media
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*Bucket with ice
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*Microcentrifuge
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*Microcentrifuge tubes
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*Micropipette
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*Disposable tips
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*200ul lysis solution
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*400ul SDS/NaOH (made fresh)
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*300ul acetate solution
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*1000ul isopropanol
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*400ul 70% ethanol
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*Kimwipes
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*100ul TE
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'''Procedure:'''
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# Inoculate 5ml of LB/antibiotic (chloramphenicol) with a single colony of transformed cells. Incubate overnight at 37ºC with shaking.
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# '''Optional''': Keep 2mL of the bacterial culture for indexing. Pipet 1.5mL of culture into a microcentrifuge tube, and microcentrifuge at 10,000 rpm for 1 minute. Discard the supernatant, and add the remaining 1.5mL of culture to the same microcentrifuge tube. Centrifuge the tube for 1 more minute at 10,000 rpm and discard supernatant one more time.
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# Resuspend the pellet in 200ul of lysis solution. Incubate at room temperature for 5 minutes.
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# Add 400ul of SDS/NaOH (made fresh) and invert 3-6 times. Incubate on ice for 5 minutes, solution will become clear and viscous.
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# Add 300ul of acetate solution, gently mix for a few seconds, and incubate on ice for 10 minutes.
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# Centrifuge for 3 minutes at 10,000 rpm in microcentrifuge.'''Optional''': Adjust microcentrifuge time depending on amount of cell growth. 
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# Decant supernatant into a clean microcentrifuge tube; avoid the loose, viscous pellet.
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# Add 1 volume of isopropanol and invert tube 3-6 times. Incubate at room temperature for 10 minutes to precipitate DNA.
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# Centrifuge at 10,000 rpm for 10 minutes. Orient hinge to the same direction in case pellet is hard to see.
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# Discard supernatant, removing residual liquid with pipet tip. Add 200-400ul 70% ethanol, agitate pellet gently and centrifuge at 10,000 rpm for 10 min to precipitate pellet.
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# Discard supernatant, being careful to avoid losing the pellet. Let rest at room temperature with the lid open or inverted on a Kimwipe to evaporate residual alcohol.
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# Dissolve pellet in just enough TE to dissolve the DNA, approx. 50-55ul. '''Optional''': Let pellet hydrate overnight.

Revision as of 23:38, 31 July 2013


Alkaline Lysis Plasmid Miniprep

Purpose: To isolate plasmid DNA from transformed cells.


Reagents:

  • Lysis solution (50mM glucose, 25mM TrisHCl pH 8, 10 mM EDTA)
9.0mL dH2O
0.25mL 1M TrisHCl pH 8
0.25mL 0.5 M Na2EDTA
0.50mL 20% glucose
  • SDS/NaOH (prepare fresh)
880ul dH2O
100ul 10% SDS
20ul 10M NaOh
  • Acetate solution
60mL 5M potassium acetate
11.5mL glacial acetic acid
28.5mL dH2O


Materials:

  • 1 colony of transformed cells
  • 5mL LB/chloramphenicol media
  • Bucket with ice
  • Microcentrifuge
  • Microcentrifuge tubes
  • Micropipette
  • Disposable tips
  • 200ul lysis solution
  • 400ul SDS/NaOH (made fresh)
  • 300ul acetate solution
  • 1000ul isopropanol
  • 400ul 70% ethanol
  • Kimwipes
  • 100ul TE


Procedure:

  1. Inoculate 5ml of LB/antibiotic (chloramphenicol) with a single colony of transformed cells. Incubate overnight at 37ºC with shaking.
  2. Optional: Keep 2mL of the bacterial culture for indexing. Pipet 1.5mL of culture into a microcentrifuge tube, and microcentrifuge at 10,000 rpm for 1 minute. Discard the supernatant, and add the remaining 1.5mL of culture to the same microcentrifuge tube. Centrifuge the tube for 1 more minute at 10,000 rpm and discard supernatant one more time.
  3. Resuspend the pellet in 200ul of lysis solution. Incubate at room temperature for 5 minutes.
  4. Add 400ul of SDS/NaOH (made fresh) and invert 3-6 times. Incubate on ice for 5 minutes, solution will become clear and viscous.
  5. Add 300ul of acetate solution, gently mix for a few seconds, and incubate on ice for 10 minutes.
  6. Centrifuge for 3 minutes at 10,000 rpm in microcentrifuge.Optional: Adjust microcentrifuge time depending on amount of cell growth.
  7. Decant supernatant into a clean microcentrifuge tube; avoid the loose, viscous pellet.
  8. Add 1 volume of isopropanol and invert tube 3-6 times. Incubate at room temperature for 10 minutes to precipitate DNA.
  9. Centrifuge at 10,000 rpm for 10 minutes. Orient hinge to the same direction in case pellet is hard to see.
  10. Discard supernatant, removing residual liquid with pipet tip. Add 200-400ul 70% ethanol, agitate pellet gently and centrifuge at 10,000 rpm for 10 min to precipitate pellet.
  11. Discard supernatant, being careful to avoid losing the pellet. Let rest at room temperature with the lid open or inverted on a Kimwipe to evaporate residual alcohol.
  12. Dissolve pellet in just enough TE to dissolve the DNA, approx. 50-55ul. Optional: Let pellet hydrate overnight.
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