(by rwmd1 from NOSPAMle.ac.uk)
Fri Jun 1 08:18:22 EST 2007
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?
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