LR cloning

Estimated time

1 hour to overnight for best efficiency

Protocol

Note

In most instances, a 5 uL reaction is sufficent for effective colonies.

  1. Add 50-150 ng of entry plasmid and 150 ng of destination plasmid. Dilute with 1X TE buffer to 4 uL or 8 uL depending on whether you choose to perform a 5 uL or 10 uL reaction respectively.

  2. Thaw LR Clonase II enzyme mix on ice for two minutes. Vortex the enzyme mix briefly (2 seconds each time).

  3. Add 1 uL/2 uL clonase mix for a 5 uL/10 uL reaction.

  4. Incubate reaction at room temperature for 1 hour to overnight.

  5. Optional, if transforming immediately: Add 0.5/1.0 uL Proteinase K to each sample to terminate the reaction. Incubate at 37 degrees C for 10 minutes.

  6. Transform competent cells with the reaction product.

Expected results

An efficent reaction will produce > 5000 colonies, if the entire volume is transformed and plated.