Toxoplasma Cosmid Libraries for Complimentation
Two separate cosmid libraries were produced from the RH (Type I) strain of T. gondii. The first of these, called pSC/Ble was constructed by Sau3a partial digestion of genomic DNA that was cloned into the BamH1 site of SuperCos from Stratagene. Prior to construction the vector was modified by insertion of a SAG-ble-SAG construct (Messina et al., 1995). The second library was made by a similar process using modified SuperCos (Strategene) that carried a DHFR-TS marker. Randomly selected clones from these libraries were end-sequenced at TIGR and the resulting sequences were mapped on to the assembled genome called ToxoDB. You can locate cosmids that span a region of interest in the genome by searching in gbrowse for T. gondii. Cosmids can be used for complimentation into T. gondii by transfection using standard methods (Roos et al, 1994).
Web based finder for locating cosmid clones created by Aliz Axmann and
Rob Bell in Jeff Chuang's lab at Boston College.
Methods and Reagents
Locating a specific clone in the 384 well plates (pdf)
Map of 384 well plates (pdf)
Cosmid prep. and sequencing protocol (pdf)
Ble transfection protocol (pdf)
Cosmid Plate List (pdf)
Toxo Cosmid transformation protocol (Streipen Lab) (pdf)
Obtaining an individual clone
Cosmids clones are available to all qualified scientists at all not- for-profit, research institutions. Commercial parties may also request materials but should keep in mind that additional paperwork will be required. Locate the clone you want in gbrowse. Send an email to "Reeha" Mary Savari Dhason. Provide the clone name, a complete shipping address, and a FedEx account number if you want rapid shipment.
|Sibley Lab (email@example.com)
Department of Molecular Microbiology
Washington University School of Medicine
St. Louis, MO USA