P.fluorescens Q8r1-96 genomic library.
We are using an ordered library of Q8r1-96 genomic DNA in the cosmid vector pCPP47(1), which contains a par locus and is stable in strain Q2-87 in the absence of selection.
This
library consists of 1536 clones with inserts averaging 30 kb in size,
and for the estimated 6-Mb genome of Q8r1-96 has a 99.9% probability of
containing a given DNA sequence. The library is suitable for both PCR-
and hybridization-based screening. For PCR screening, the library was
divided into 16 "primary" pools (i.e. all clones from a defined
microtiter plate) that were further divided into a series of "secondary"
pools (containing all clones in the same row or column from a defined
microtiter plate). Positive primary pools are subsequently analyzed by
row and column PCR, enabling us to pinpoint specific clones.
For screening by hybridization, the library was arrayed on sets four,
7.4 x 11.4-cm nylon membranes in a 386-sample format that allows one to
screen 1536 clones (4 membranes) in a single experiment. The clones were
replicated manually from glycerol stocks onto membranes with a 96-pin
Multi-Blot Replicator (1.58 mm diameter solid pins, 0.2 ml delivery per
sample) (V&P Scientific, Inc., San Diego, DA) and a library copier
that permits arraying in a 386-sample format. These membranes can be repeatedly
hybridized with 32P-labeled probes.
References
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Bauer, D. W., and A. Collmer. 1997. Molecular cloning, characterization, and mutagenesis of a pel gene from Pseudomonas syringae pv. lachrymans encoding a member of the Erwinia chrysanthemi pelADE family of pectate lyases. Mol. Plant-Microbe Interact. 10:369-379.