Team:Alberta/Notebook/protocols/transformations

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Revision as of 00:38, 27 October 2010

TEAM ALBERTA

Transformations

Reagents:

  • Competent DH5α cells (100uL per transformation)
  • up to 5uL of plasmid you want to transform
  • 800uL of LB (non-contaminated) per transformation
  • Plate with correct antibiotics


Procedure:

  • Chill a box of pipette tips at -4oC and put your reaction tubes on ice.
  • Obtain DH5α cells from the –80oC freezer and thaw in palm. Store on ice for 10 minutes.
  • Use a chilled tip to transfer 100 ul of the DH5α cells into each tube. Set aside one tube as a control.
  • Pipet up to 5uL of the plasmid (up to 50ng per 100ul of competent cells, plasmid amount. Should not exceed 5% that of the competent cells) into each tube. Swirl.
  • Store on ice for 30 minutes.
  • Place tubes in incubator set to 42oC for EXACTLY 90 seconds.
  • Return the tubes to ice for 2 minutes.
  • Add 800uL of LB to each tube.
  • Incubate at 37oC for 45 minutes, ensure that the tube has at least 2ml capacity, to properly aerate the cells.
  • Spread cells on plate with appropriate antibiotic: plate between 50- 300ul each. Let dry.
  • Place plates inverted in incubator at 37oC for 12-16 hours.


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