(by peter.ianakiev from gmail.com)
Fri Jun 1 09:13:32 EST 2007
On Jun 1, 9:18 am, Astala Vista <r... from NOSPAMle.ac.uk> 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?
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,
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