About ligation

vilimf01 at mcrcr6.med.nyu.edu vilimf01 at mcrcr6.med.nyu.edu
Sun Apr 21 11:43:01 EST 1996


In article <drm21-2004961617450001 at nntp-serv.cam.ac.uk>, drm21 at mole.bio.cam.ac.uk (David Micklem) writes:
> 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.


If this is true, one would want to do blunt ligations at 37 where the
ligase is most active.  Is this true?  Has anyone out there tested
this?

			Regards			SVEN








> 
> 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,              
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> Tel: [+44] (0)1223 334129     Email:drm21 at mole.bio.cam.ac.uk
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