eettehad at direct.ca
Sun Apr 21 19:01:22 EST 1996
In article <960419181331.42212c84 at chemvx.tamu.edu>, KHUANG at CHEMVX.TAMU.EDU
> Hi, Netters,
> I have a few silly question to ask you.
> (1) About ligation temperature.
> Any enzyme (?) should have its best activity temperature. Some protocol
> said we can do ligation at 4, 16, 22-25 degree, It is said T4 DNA ligase
> has a few best activity temperature, right?
> (2) About the size of insert
> It is difficult to do ligation for longer insert. For pBluescript, how
> longest insert can be ligated (I heard of 21 kb)? Do you think it is
> possible to find (or creat) new ligase for blunt, long insert ligation et al?
> (3) What is the secret in the 30 min ligation kit? New ligase or inducer
> exist there?
here is some wisdom for you,
from my experience the temp depends on what kind of ligation you want to
If its a hard one to get ligated, like blunt, three way ligation..etc then
do it over night at 15 C. If its an easy one, sticky end and stuff then
just room temp for couple of hours should do the job. Also if you want to
leave any kind of ligation over night then defenatley put it at 15 C
because thos pesky Dnases can get to your DNA and damage the ends really
badly if its at high Temps.
and by the way I always use T4 DNA ligase from NEB. Another thing is that
if your ligation is a difficult one add couple of microliters of 40% PEG in
there, it helps a lot.
as far as size the largest one I have been able to clone into a PGEM
plasmid was 12 kb, it was a bit tricky but it worked..again I had lotsa
insert and it was left at 15 C overnight.
Have never ever used a kit for a ligation! it really is not
neccesary...trust me, I have done the hardest ligations without a kit.
Just some T4 ligase and its buffter and a bit of PEG can do the job.
Good luck!! and happy cloning!!!
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