Dear Chromosome Group,
We are investigating a small algal chromsome believed to carry genes
involved in photosynthesis. The chromosome has an apparent size of
95kb in pulsed field gels. Would it be feasible to clone the entire
chromosome into bacteriophage P1? We would welcome comments on the
following strategy.
If the ends of the 95kb chromosome are typical of eukaryotic
chromosomes, they will contain telomeres with short 3' overhangs.
These overhangs could be trimmed by the 3'-5' exonuclease activity of
the T4 polymerase. Dephosphorylated BamH1 adaptors could then be
ligated to the ends of the chromosome and the adaptors phosphorylated
to allow the adaptor/chromosome complex to be ligated into the
dephosphorylated site in the vector.
We have chromosome-specific probes and STS PCR primers to screen for
inserts.
We envisage two possible DNA preps as start points.
1/ Cutting the 95kb band out of a pulsed field gel and using agarase
to get the chromosome out intact.
2/ Alternatively we could use total high molecular weight genomic
DNA (>200kb) from the algal cells. The size selection would then be
impemented by the phage packaging system.
Does anybody have any experience, opinions, pitfalls to avoid. Is
this feasible in your experience. We keep telling ourself that we
only need one clone, not a comprehensive library.
Thanks
Geoff McFadden and Paul Gilson
Plant Cell Biology Research Centre
University of Melbourne, Parkville 3052 VIC
Australia
_--_|\ email: u6065606 at ucsvc.ucs.unimelb.edu.au
/ \ telephone: +61 3 344 5062
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