I am no expert but I have performed several Gateway Cloning reactions and almost always got a lot of colonies. Here are some suggestions that you can try.

-Prepare your entry clone in TE buffer (pH 8.0)

-Use more entry clone (I usually make entry clone to destination vector in the ratio 2:1)

-Vortex the LR Clonase II enzyme (2µl) before adding to the reaction mixture

-Incubate the mixture at 25°C for 1hr and at 4°C overnight (optional)

-After the reaction is complete, add 1µl Proteinase K solution, vortex briefly and incubate at 37°C for 10min

You can now transform the cells. I always take One Shot TOPO10 cells and add 1µl of the reaction mix and use heat shock method (30 min in ice; 30sec at 42°C) and then add 250µl SOC medium; incubate at 37°C in a shaker for 1-2 hr and then finally to selective agar plates. Make sure to choose the right antibiotic.

I have also tried with half the reaction mixture (5µl) which also worked fine. You will only need 1-2µl for transformation.

All the best!

Agreeing with all Saroj Pandey said, I would stress the proteinase k treatment. I've tried in parallel with and without proteinase k treatment and it was almost 10x more colonies with!

I would also check the competency of your cells. That can also make a difference...

Best regards

Saroj offers good advice. I thought I'd offer some practical advice. If you get at least one colony that has your cloned sequence, then that's all you need. The efficiency doesn't have to be amazingly high as long as you get what you need.

Thank you all. Hope it will help me a lot.