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