>>For two months now I have been trying to clone a 2 kb blunt fragment
>(NruI/HindIII, filled in with klenow) into a PvuII site of a 6.5 kb
>>2. From the catalog a learned that blunt NruI sites are difficult to
>ligate. Does anybody know why, and if there is a way to imporve this?
>>>Have you considered the isoschizomer Bsp68I? Maybe NruI is simply one of
>those not very reliable enzymes, hard to get very pure, or something.
>SmaI used to have a bad reputation like this (Waaay back when).
the enzyme is available from:
Angewandte Gentechnologie Systeme (REBASE abbr: D)
Rischerstrasse 12, W-69123 Heidelberg
according to REBASE. Also from Fermentas, which trades through Biolabs,
I routinely use blunt-blunt cloning, and it works fine, with just the
normal DNA ligase (though I use a full unit per ligation); you *must*
have a functioning vector, though! ;-)
If you *have* to use Nru, I would polish the ends with T4 DNA pol, as
this is better than klenow at 3' overhangs, and you don't know what the
enzyme is doing to your DNA.
Contact me if you need any more help.
michael.baron at bbsrc.ac.uk