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Methods for Genome Mapping
In the Filarial Genome Project we have chosen a mixed strategy of mapping the genome using cosmid, BAC and YAC libraries by hybridisation (see here for a discussion of the rationale for this). We are collating and testing genome mapping methods for the project. Many of these are derived from other workers, and all have been adapted to improve their efficiency or to suit our needs better. They are listed here for use by others in the genome project and elsewhere.
For generation of end-probes from BACs, we
have developed a PCR based system for getting the last 300-500 bases
of a BAC.
Edinburgh BAC end-probe method (coming soon)
For mapping using cDNA clones as probes, the mapping process uses the same clone preparation and labelling methods as we use for cross-screening the cDNA clones:
Construction of the multipronged "hedgehog" device for colony replicationMaking replica gridded filters using a multipronged "hedgehog" device
We have chosen to perform our hybridisation screening using nonradioactive methods, to make it easier for endemic country labs to use them.
Making probe DNAs using PCR from plaques or plasmidspBluescript sequence map showing PCR primers
New England Biolabs "Phototope" Labelling and detection
Amersham "ECL" Gene Images labelling
for the record, here is the Radioactive Labelling Protocol
Preparation of large-insert clone DNA is slightly more difficult than standard plasmid preparation
Edinburgh BAC preps (coming soon)
