Team:Brown-Stanford/Lab/Protocols/FastCaCl

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<html><h1>Protocol Code: CT4</h1></html>
<html><h1>Protocol Code: CT4</h1></html>

Revision as of 17:39, 27 September 2011

Brown-Stanford
iGEM
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Protocol Code: CT4

Fast CaCl2 Combined Competency and Transformation

Required Solutions

  • 2.5mL of 50mM CaCl2
  • 5.5 mL liquid LB media

Competency

  1. Take cell culture and grow in 5mL LB liquid media overnight until OD600 = 0.6-0.7.
  2. Cool cells on ice for 10 minutes.
  3. Centrifuge cells at 3000rpm, 4oC, for 5 minutes.
  4. Discard supernatant and place cells back on ice.
  5. Resuspend cells in 2mL of cold CaCl2 (50mM).
  6. Cool cells on ice for 15 minutes.
  7. Centrifuge cells at 3000rpm, 4oC, for 5 minutes.
  8. Discard supernatant and place cells back on ice.
  9. Resuspend cells in 500µL of cold CaCl2 (50mM).

Transformation

  1. Take 100-200µL of the resuspended cells and place in a precooled microfugetube.
  2. Add DNA ligation mixture.
  3. Incubate on ice for 20-30 minutes. Prepare 42oC water bath.
  4. Heat shock cells in water bath for 2 minutes.
  5. Move cells back onto the ice.
  6. Add 0.5mL LB media.
  7. Incubate cells at 37oC for 1 hour.
  8. Spread cells over 1-2 LB/antibiotic plates, and invert in incubator overnight.

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