My first suggestions:

a) check the buffer for the reaction which contains ATP. Make sure to use fresh one and avoid freeze and thawing cycles.

b) quality of competent cells? Do you use bought competent cells or made by yourself, electro-competent or chemical-competent?

However, 10 colonies are okay, since you need only one correct clone. Or do you want to prepare a library?

Regards, Marlen

My first suggestions:

a) check the buffer for the reaction which contains ATP. Make sure to use fresh one and avoid freeze and thawing cycles.

b) quality of competent cells? Do you use bought competent cells or made by yourself, electro-competent or chemical-competent?

However, 10 colonies are okay, since you need only one correct clone. Or do you want to prepare a library?

Regards, Marlen

I based my PhD on gateway cloning. Actually, the amount of colonies is irrelevant. The important issue is that the colonies you are obtaining are containing the right plasmid. I succesfully cloned 3-4 kb fragments obtaining 2-3 colonies (all positive!!!!)

Not getting too many colonies is not an issue. A single positive clone would help. Your insert size is big (3.7kb), what is the vector size, try ligation of vector:insert in 1:1 ratio.

have you checked the colonies for clone confirmation?

Have you checked the transformation efficiency of competent cells.

I've done a lot of Gateway cloning and never got the quantity of colonies the instructions imply. But the efficiency of getting the correct construct was at least 75% in my hands, so as mentioned before, a low number of colonies is ok. That being said, I always did at least 2 hour incubation at 25 deg, and plated different amounts of the transformation mix, (10%, 30%, 60%) on the selective plates.