Double insert ligation? - (Jun/03/2005 )

Hi!

I was wondering if it is possible to do a cloning experiment where you insert 2 fragments into your vector using different enzymes? It seems highly unlikely that you can do this and if so, probably very tricky.

But I would appreciate some advice on whether there is such a protocol or if I should just insert one fragment, miniprep, and then insert second fragment.

Thanks!

hi
all is feasible in theory. If you can do a sequential insertion, i would say it's a better way. But once a colleague done two insertions and after a great number of PCR based screen, she found her right colony.
i woul try it as a personal challenge.

In my hands, the efficiency is 10-fold less for a 3-way ligation. For example, instead of 300 colonies per plate, I get 30.