Standard Protocols

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(Transformation into Competent Cells)
(Transformation into Competent Cells)
Line 13: Line 13:
# Add 1 ml of LB liquid media
# Add 1 ml of LB liquid media
# Shake for about an hour at 37°C
# Shake for about an hour at 37°C
-
# Plate on agar containing the correct antibiotic
+
# Plate on agar containing the correct antibiotic, put approx. 100ul of cells on one plate, and the remainder on the other after centrifuging and resuspending the pellet
# Incubate at 37°C overnight
# Incubate at 37°C overnight
# Remove from incubator and rest at room temperature for approximately an hour
# Remove from incubator and rest at room temperature for approximately an hour

Revision as of 13:59, 6 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, put approx. 100ul of cells on one plate, and the remainder on the other after centrifuging and resuspending the pellet
  8. Incubate at 37°C overnight
  9. Remove from incubator and rest at room temperature for approximately an hour
  10. Move to refrigerator


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