LambdaGem-11 cloning problems

[Susanne Rafelski]

Hi All!

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
inversion
gel electrophoresis). This showed that the genomic DNA preps were of
high
molecular weight, much of the genomic DNA being above 75 KB.

The genomic DNA was partially digested with MboI and the fragment of
15-23
kb was isolated from a FIGE gel and electroeluted using Schleicher and
Schuell's Elutrap system. The obtained band was checked once again on
FIGE
and seemed to not be degraded. Control ligation of insert provided in
the
kit to the vector arms produced phage  with titer of 10^6 pfu/ug vector
as
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
KBto
the  vector arms did not produce any plaques beyond the background
plaques.
The ligation was checked  on FIGE, and the bands representing the vector

arms as well as the insert genomic DNA diminished in concentration, and
a
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
sees a
band that is the size of both arms together, probably via the Cos sites
at
the ends of the vector that are not being used for cloning. Thus if
insert
DNA that was only cut with MboI at one end  ligated to  this fragment,
the
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
at
least some of the insert can ligate at both ends,

our opinion is that possibly not enough of the insert DNA is digested
with
MboI on both ends so it sucks up the available vector arms in the
ligation,
hence our ligation gel results, but does not produce packagable vector
that
would then produce phage.

Do you have any ideas, suggestions on how we could proceed?
Any help will be greatly appreciated

Susanne Rafelski
Institute of Plant Molecular Biology
Ceske Budejovice
Czech Republic