IUBio Biosequences .. Software .. Molbio soft .. Network News .. FTP

Rapid Ligation Kit

Frank O. Fackelmayer Frank.Fackelmayer at uni-konstanz.de
Thu Jan 6 04:14:21 EST 2000


Bruno Cenni wrote:

> We do the short version also quite often (blunt and sticky ends ligations in low
> melt agarose snippets) and it works (almost) as well as the typical overnight
> ligation (using Life Techn T4 Ligase). If I remember well the Boehringer kit simply
> uses high amounts of ligase and a buffer containing PEG or something like that.
>
> Bruno
>
> >
> > With sticky end ligations, I've been able, on more than one occasion, to
> > prep vector and insert, add standard ligation buffer and ligase, incubate
> > on the bench (room temp) for 30 mins to an hour, electroporate, and be
> > looking at colonies the next morning.

Hi Bruno and David,
Even though ligation for a short time works in some cases, I wouldn´t recommend it as
standard procedure. I used to ligate for 4h at RT for most of my clonings, and did get
clones in most cases. However, when I recently did a direct comparison between 30min,
4h and 16h ligations (withdrawing aliquots from the same reaction), I got several
hundred colonies in th 16h assay, around 20 in the 4h assay, and only 1 in the 30min
assay. Without doing sophisticated statistics, it is obvious that a longer ligation
time is beneficial for getting a higher number of colonies. For a simple cloning
(where one positive clone is already the optimal result) it is certainly not necessary
to have hundreds of clones of which you analyse, say, some 20 only. In other cases,
however, you will need a good ligation efficiency. As it is sometimes difficult to say
if a particular cloning will be simple or not, I´d go for overnight ligation on a
routine basis. It actually SAVES time in comparison to short ligations that don´t
work   : )

Of course, if you are in a hurry and you may be happy with the short ligation.
Remember, however, that not only ligation efficiency counts, but also transformation
efficiency. So, use really good competent cells for all clonings (> 10e8 transformants
per ug of supercoiled plasmid), especially when you cut down ligation time. I
personally like electroporation because it never fails (well....)

Frank






More information about the Methods mailing list

Send comments to us at biosci-help [At] net.bio.net