LambdaGem-11 cloning problems
susanner at u.arizona.edu
Wed Jun 30 06:34:15 EST 1999
We are attempting to make a genomic Library from Pea, using the Promega
LambdaGem-11 BamHI arms kit.
We isolated genomic DNA and checked its quality using FIGE (field
gel electrophoresis). This showed that the genomic DNA preps were of
molecular weight, much of the genomic DNA being above 75 KB.
The genomic DNA was partially digested with MboI and the fragment of
kb was isolated from a FIGE gel and electroeluted using Schleicher and
Schuell's Elutrap system. The obtained band was checked once again on
and seemed to not be degraded. Control ligation of insert provided in
kit to the vector arms produced phage with titer of 10^6 pfu/ug vector
expected from the kit handbook. ligation of only the vector arms showed
that the background was low, about 10-50 pfu/ug, again as expected.
Ligations of the purified (and quantitated) genomic DNA of size 16-23
the vector arms did not produce any plaques beyond the background
The ligation was checked on FIGE, and the bands representing the vector
arms as well as the insert genomic DNA diminished in concentration, and
smear upwards was seen, so nothing was preventing the ligation. In fact
these results should lead to plaques after the ligations are packaged.
One interesting thing is that when running the vector arms only, one
band that is the size of both arms together, probably via the Cos sites
the ends of the vector that are not being used for cloning. Thus if
DNA that was only cut with MboI at one end ligated to this fragment,
smear would be in the size range that would normally demonstrate the
existance of both vector arms ligated to insert.
Self ligation of the insert was also checked on FIGE, and showed that
least some of the insert can ligate at both ends,
our opinion is that possibly not enough of the insert DNA is digested
MboI on both ends so it sucks up the available vector arms in the
hence our ligation gel results, but does not produce packagable vector
would then produce phage.
Do you have any ideas, suggestions on how we could proceed?
Any help will be greatly appreciated
Institute of Plant Molecular Biology
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