Gateway Cloning

Overview

Read the Gateway cloning manuals from Invitrogen for a more detailed version of this protocol

Protocol

L-R Clonase reaction Using LR Clonase Enzyme mix kit

  • Add the following to a 1.5 ml microcentrifuge tube at room temp:
    • 300 ng of Entry clone
    • 300 ng of Destination vector
    • Note: Do not use more DNA as it could create colonies with multiple DNA molecules, which is associated with the "small colony" phenotype
    • 4 µl of 5x LR Clonase Reaction Buffer
    • up to 16 µl TE buffer, pH 8.0
  • Remove LR Clonase enzyme mix from -80 and thaw on ice for about 2 min.
  • Vortex LR Clonase enzyme mix briefly 2x for 2 seconds
  • Add 4 µl of LR Clonase to the microcentribfuge tube and vortex briefly 2x for 2 seconds
  • Spin tube briefly
  • Return LR Clonase enzyme mix to -80 immediately
  • Incubate reaction 1 hr to overnight at RT.
  • Add 2 µl Proteinase K vortex briefly, and incubate 37ºC for 10 minutes.
  • Transform using CaCl2 transformation.

L-R Clonase reaction Using LR Clonase II enzyme mix

  • Add the following to a 1.5 ml microcentrifuge tube at room temp:
    • 150 ng Entry clone
    • 150 ng Destination vector
    • up to 8 µl TE buffer, pH 8.0
  • Remove LR Clonase II enzyme mix from freezer and thaw on ice for about 2 min.
  • Vortex LR Clonase II enzyme mix briefly 2x for 2 seconds
  • Add 2 µl of LR Clonase II enzyme mix to the microcentribfuge tube and vortex briefly 2x for 2 seconds
  • Spin tube briefly
  • Incubate reaction 1 hr to overnight.
  • Add 1 µl Proteinase K vortex briefly, and incubate 37ºC for 10 minutes.
  • Transform using CaCl2 transformation.