Ligation artefacts

peter via (by peter.ianakiev from
Fri Jun 1 09:13:32 EST 2007

On Jun 1, 9:18 am, Astala Vista <r... from> wrote:
> We recently constructed a small human genomic DNA library in a plasmid
> vector for use in an undergraduate practical class. The human DNA was
> partially digested with Sau3AI and was size selected for fragments
> between 2 kb and 4 kb. The plasmid was pUC18NotI which has NotI sites
> flanking the normal pUC18 MCS. The vector was digested to completeion
> with BamHI and was then phosphatased. Ligations were set up and DNA was
> electroporated into E. coli DH5alphaE. Colonies were picked at random
> and mini-prepped. Around 100 random clones were sequenced from each end
> of the insert using primers located far enough back that we could read
> through the plasmid/insert junction.
> As you would expect, a minority of junctions recreated the BamHI site,
> and the majority only created a Sau3AI site. However, 7 of the junctions
> had neither a BamHI site or a Sau3AI site. Several bases had been
> altered in the vicinity of the jucntion.
> Has anybody seen such a high level of ligation problems before? Any
> guesses about what's going on?
> Thanks.

It is not unusual, likely artifacts come from long and strong CIP
incubation (use 1:10 dilution in RE buffer of the CIP, and incubate
for not more than 10-15 min at RT). Also it could be that you have
some nuclease contamination, probably form your reagents / buffers
(ligase most likely). Last point I would like to make - do not use too
much of the vector and the insert, 10ng of each should do it, and do
not transform more than 1ul per reaction.
Hope this helps,

More information about the Methods mailing list