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

-oetziline-

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

-casio-

QUOTE (casio @ Apr 27 2007, 10:25 AM)

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

-oetziline-

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

-perneseblue-