# Cloning of synthetic oligos - how much oligos for ligation? (Apr/26/2007 )

Hi,

I'm going to clone a 120bp promoter fragment into the pGL3 vector. I'm going to use synthetic oligos which are already tailed with the sticky ends of two restriciton enzymes (KpnI & MluI). I'm wondering how much of these oligos I should anneal and how much of the annealing mixture I should use for the ligation (and how much vector DNA).

Has anyone experience with cloning of such long synthetic oligos?

Thanks for any help!

Carolin

Hi Carolin

I suppose first you have to dilute them to 10pm/microlitre. Mix them together and phosphorylate by PNKinase. Then you ligate them with vector in 3:1, 4:1 ratios. I am sure it will work.

Cheers

I suppose first you have to dilute them to 10pm/microlitre. Mix them together and phosphorylate by PNKinase. Then you ligate them with vector in 3:1, 4:1 ratios. I am sure it will work.

Cheers

Thanks! I'm wondering if I really need to phosphorylate them since the vector will be phosphorylated. That should be sufficient, right?

yes it would be okay. However depending on the ligation strategy you may have to fight through lots of self ligated vector , if the vector ends are compatible. None compatible ends produce far fewer self ligated molecules, but you will still see a fair few.

The strategy outline previously (with phosphorylated oligoes) suffers from a significant rate of concentamer formation. The 120bp fragment will to a degree concentamerise (depending on insert concentration and structure-my suspicion), and insert into the vector. You will then have to screne against these concentamers.

So you have a choice of poisons