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.

Warning

LR clonase does expire and you’ll get weird fragments or the unreacted backbone. Make sure to pay attention to the expiration date (~1 year)