Gateway BP Clonase II Reaction

Note: This protocol is NOT compatible with BP Clonase (it is specifically for BP Clonase II).

Contents

1. Overview
2. Materials
3. Procedure

1. Overview

Protips: The pEXP7-tet fragment is a tetracyclin resistance gene flanked by attB sites. It comes with the Clonase kit and can be used as a positive control. Stoichiometry of DNA is important and significantly affects the efficiency of the reaction. The reaction must be run for at least one hour which should suffice for most purposes, however, it can run as long as 18 hours. Running it for 18 hours yields 5-10 times as many colonies as a one-hour reaction. It is important that your DNA fragment and pDONR plasmid be concentrated enough that your total reaction volume remains below 10µL, as this can affect efficiency. There’s a lot of glycerol in these reactions so vortexing is important.

Detailed manual: <http://tools.lifetechnologies.com/content/sfs/manuals/gateway_clonaseii_man.pdf PDF>

2. Materials

• Sample: attB-flanked linear gene fragment (150ng)
• Entry clone: pDONR221 (150ng per sample)
• TE Buffer, pH 8.0
• Positive control: pEXP7-tet (2µl)
• BP Clonase II
• Proteinase K (comes with BP Clonase II kit)
• Competent DH5α

3. Procedure

1. Add to a 1.5mL microcentrifuge tube:
   • 150 ng sample
   • 150 ng entry clone
   • TE, to 8µL
2. Thaw BP Clonase II on ice for 2 minutes.
3. Vortex BP Clonase II twice for 2 seconds.
4. Add 2µL BP Clonase II to each sample.
5. Put the BP Clonase II back in the -80°C freezer.
6. Vortex each sample for a few seconds and use a mini centrifuge to bring the liquid back to the bottom of the tube.
7. Incubate at 25°C for 1 to 18 hours (see protips above).
8. Add 1µL Proteinase K to each sample.
9. Incubate for 10 minutes at 37°C.
10. Transform into DH5α and plate on LB-Kanamycin plates (or LB-Tetracycline plates for the positive control).