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| | {{:Team:Glendale_CC_AZ/test}} | | {{:Team:Glendale_CC_AZ/test}} |
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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.
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