About ligation

David Micklem drm21 at mole.bio.cam.ac.uk
Sat Apr 20 11:17:45 EST 1996


In article <960419181331.42212c84 at chemvx.tamu.edu>, KHUANG at CHEMVX.TAMU.EDU
wrote:

>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?

Hi Huang,
     I suspect that the optimal temperature for DNA ligase is 37C, since
T4 is a bacteriophage, and is likely to like the same temperature that its
host likes. 
    The temperature we do ligations at is a compromise between ligase
activity and DNA annealing: at low temperatures, the short overhangs we
are usually trying to ligate are more likely to be stuck to each other
than at high temperatures, but the ligase will be less active.

Personally, I do all my ligations at room temperature and they work fine
(see below).

>(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?

I don't think that the problem here is in the ligation - I don't think the
ligase particularly cares how long a piece of DNA the end it is acting on
is attached to! I suspect its more a problem of the host cell not
tolerating very large, high copy number plasmids.   There might be some
potential for a better blunt-end ligator - anything that encourages the
DNA ends to spend more time in close proximity (eg PEG) should help.  

>(3) What is the secret in the 30 min ligation kit? New ligase or inducer 
>exist there?

As far as I can tell, there IS no secret ingredient - ligations done with
regular NEB ligase go to completion very quickly at room temperature. 
Someone posted here a while ago the results of a time course for blunt-end
ligation that agreed very well with my own impression that there is very
little difference between 1hr and overnight.  For sticky-end ligations,
I've always found 15 minutes to be sufficient.

Cheers,

David

_____________________________________________________________
D.R.Micklem,
Wellcome/CRC Institute,       Time flies like an arrow...
Tennis Court Road,              
Cambridge CB2 1QR             Fruit flies like a banana.
UK                             
Tel: [+44] (0)1223 334129     Email:drm21 at mole.bio.cam.ac.uk
Fax: [+44] (0)1223 334089               
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