Ligation problems

wmbonner at helix.nih.gov wmbonner at helix.nih.gov
Thu Mar 16 21:10:31 EST 1995


In article <D5FG0n.1qM at demon.co.uk>, Anthony Tomlinson <tomlinson at pplros.demon.co.uk> wrote:
>In article <3js36s$e27 at thot.u-strasbg.fr> Francois Nantel,
>Nantel at titus.u-strasbg.fr writes:
>>Dear bionetters,
>>
>>I presently have big ligation problems but that was expected. I need to
>>put a 3kb insert into a 3.2 kb vector. Oh, did I tell you it is a blunt
>>ligation :-0
>>
>>I tried treating the vector with alkaline phosphatase but the ligation
>>effeciency is so low I didn't get anything.
>>
>>If anybody have a knock-out technique for hard to do ligation, I would be
>>very grateful.
>
>Last time I had a hard blunt to do, I tried Ready to Go dried down ligase
>from
>Pharmacia, and it worked a treat.  It's not cheap, but they sent me a
>free sample which was enough for
>my experiment!
>
>Anthony

I've had good luck with the pCR-script kit from Stratagene.  It uses a rare 8-base cutter (I forget 
which) to recut the vector each time it closes without the insert.  No need to dephos. Good as long 
as your frag does not have the 8-base cutter site.
-Bill



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