Standard Protocols

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# Place aliquot in heat block set to a temperature of 42°C for approximately 2 minutes (no longer than 3 minutes)
# Place aliquot in heat block set to a temperature of 42°C for approximately 2 minutes (no longer than 3 minutes)
# Return to ice for a short period of time (approximately 3 minutes)
# Return to ice for a short period of time (approximately 3 minutes)
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#  
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# Add 1 ml of LB liquid media
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# Shake for about an hour at 37°C
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# Plate on agar containing the correct antibiotic
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# Incubate at 37°C overnight
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and transform into your favorite competent cells, plate out on a plate with the correct antibiotic and grow overnight. Your goal here is to obtain single colonies.
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# Remove from oven and rest at room temperature for approximately an hour
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      4. Pick a single colony and inoculate some broth (with the correct antibiotic) and grow ~18 hours.
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# Move to refrigerator
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      5. Use the resulting culture to miniprep AND make your own glycerol stock (for further instruction on making a glycerol see this page).
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Revision as of 15:22, 5 July 2006

Resuspension of dry DNA

  1. Puncture a hole through the foil with a pipette tip
  2. Add 15 ul of distilled water

Transformation into Competent Cells

  1. Take 1ul of suspended DNA and add to an aliquot of competent E-Coli cells (taken from the freezer at -80°C)
  2. Mix gently and leave in ice bucket for 10 to 30 minutes
  3. Place aliquot in heat block set to a temperature of 42°C for approximately 2 minutes (no longer than 3 minutes)
  4. Return to ice for a short period of time (approximately 3 minutes)
  5. Add 1 ml of LB liquid media
  6. Shake for about an hour at 37°C
  7. Plate on agar containing the correct antibiotic
  8. Incubate at 37°C overnight
  9. Remove from oven and rest at room temperature for approximately an hour
  10. Move to refrigerator
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