2 ways to improve it

Benedict Chambers bencha at ki.se
Tue Feb 8 09:58:18 EST 2000




> Dear Caroline,

a) We have optimized following buffer for blunt end ligations. It improves
the ligation efficiency 10-100 fold over the buffer supplied with the NEB
and Boerhinger T4 DNA ligase. It has revolutionized our blunt-end clonings
in the lab.
Make 5x BLi buffer as following:

Salt stock: 330 mM Tris-Cl pH 7.4, 50 mM MgCl2, 50 mM DTT
To 500 µl salt stock add: 150 µl 5M NaCl, and 0.5 g PEG 8000
It will become 1 ml. Can be stored for > 6 months at -20°C.

Use at a 5-time dilution at 16°C ligations using 2 units of ligase. Before
use, add 0.5 mM ATP.

b) Reduce your background by destroying self-ligated vector as well as
never-linearized vector by digesting your ligation reaction with SmaI prior
to the transformation step. Of course, you will only be able to do this if
your desired construct lacks a SmaI site.

5 µl post-ligation mix
5 µl 10x RE buffer of your choice
40 µl aq
enzyme
Room temp 30 minutes
phenolize, precipitate, wash etc as usual
resusp in 5 µl aq

You will notice the difference!

Good luck
Peter Berglund
Peter.Berglund at mtc.ki.se







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