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Team:Glendale CC AZ/igem.org
From 2013.igem.org
(Difference between revisions)
| Line 69: | Line 69: | ||
Procedure: | Procedure: | ||
# Inoculate 5ml of LB/antibiotic (chloramphenicol) with a single colony of transformed cells. Incubate overnight at 37ºC with shaking. | # Inoculate 5ml of LB/antibiotic (chloramphenicol) with a single colony of transformed cells. Incubate overnight at 37ºC with shaking. | ||
| - | # | + | # '''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. |
# Resuspend the pellet in 200ul of lysis solution. Incubate at room temperature for 5 minutes. | # Resuspend the pellet in 200ul of lysis solution. Incubate at room temperature for 5 minutes. | ||
# Add 400ul of SDS/NaOH (made fresh) and invert 3-6 times. Incubate on ice for 5 minutes, solution will become clear and viscous. | # Add 400ul of SDS/NaOH (made fresh) and invert 3-6 times. Incubate on ice for 5 minutes, solution will become clear and viscous. | ||
# Add 300ul of acetate solution, gently mix for a few seconds, and incubate on ice for 10 minutes. | # Add 300ul of acetate solution, gently mix for a few seconds, and incubate on ice for 10 minutes. | ||
| - | # Centrifuge for 3 minutes at 10,000 rpm in microcentrifuge. | + | # Centrifuge for 3 minutes at 10,000 rpm in microcentrifuge.'''Optional''': Adjust microcentrifuge time depending on amount of cell growth. |
# Decant supernatant into a clean microcentrifuge tube; avoid the loose, viscous pellet. | # Decant supernatant into a clean microcentrifuge tube; avoid the loose, viscous pellet. | ||
# Add 1 volume of isopropanol and invert tube 3-6 times. Incubate at room temperature for 10 minutes to precipitate DNA. | # 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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# 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. | # 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. | ||
# 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. | # 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. | ||
| - | # Dissolve pellet in just enough TE to dissolve the DNA, approx. 50-55ul. Optional: Let pellet hydrate overnight. | + | # Dissolve pellet in just enough TE to dissolve the DNA, approx. 50-55ul. '''Optional''': Let pellet hydrate overnight. |
Revision as of 06:09, 30 July 2013
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:
- Inoculate 5ml of LB/antibiotic (chloramphenicol) with a single colony of transformed cells. Incubate overnight at 37ºC with shaking.
- 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.
- Resuspend the pellet in 200ul of lysis solution. Incubate at room temperature for 5 minutes.
- Add 400ul of SDS/NaOH (made fresh) and invert 3-6 times. Incubate on ice for 5 minutes, solution will become clear and viscous.
- Add 300ul of acetate solution, gently mix for a few seconds, and incubate on ice for 10 minutes.
- Centrifuge for 3 minutes at 10,000 rpm in microcentrifuge.Optional: Adjust microcentrifuge time depending on amount of cell growth.
- Decant supernatant into a clean microcentrifuge tube; avoid the loose, viscous pellet.
- Add 1 volume of isopropanol and invert tube 3-6 times. Incubate at room temperature for 10 minutes to precipitate DNA.
- Centrifuge at 10,000 rpm for 10 minutes. Orient hinge to the same direction in case pellet is hard to see.
- 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.
- 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.
- Dissolve pellet in just enough TE to dissolve the DNA, approx. 50-55ul. Optional: Let pellet hydrate overnight.
