Protocol/1

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Revision as of 00:26, 26 October 2010

TEAM ALBERTA

Protocol 1: Transformations


What you will need:

  • Competent DH5α cells (50 uL per transformation)
  • 2 uL of plasmid you want to transform
  • 800 uL of LB (non-contaminated) per transformation
  • Plate with correct antibiotics


Procedure:

  • Chill a box of pipette tips at -4 C and put your reaction tubes on ice.
  • Obtain DH5α cells from the –80 C freezer and thaw in palm. Just as they thaw, store on ice for 10 minutes.
  • Use a chilled tip to transfer 50 ul of the DH5α cells into each tube. Set aside one tube as a control.
  • Pipet 2uL of the plasmid (up to 25ng per 50 ul of competent cells, plasmid amt. 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 42 C for EXACTLY 90s.
  • Return the tubes to ice for 2 min.
  • Add 800uL of LB to each tube.
  • Incubate at 37 C for 45 min, 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 37 C for 12-16 hours.


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