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Experts present methods and protocols essential for understanding parasites at the molecular level. The protocols cover culturing techniques for the major experimental organisms, methods for isolating and processing nucleic acids and proteins, PCR-based protocols for parasite identification, gene isolation and mutation, antibody-based procedures, chromosome and epitope mapping, flow cytometry, RNA sequencing, and parasite transformation.
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Culturing and Biological Cloning of llypanosoma brucei Mark Carrington 1. Introduction Any biochemical analysis is usually made easier by the availability of large numbers of cells to be analyzed, and one of the reasons for the position held by Trypanusoma brucei as the best characterized parasite is the relative ease with which it can be cultured in the laboratory. The ability to culture cloned populations derived from individual trypanosomes before and after an antigenic switch is vital in investigations into the mechanism of antigenic variation. Genomic DNA prepared from such cloned populations used to analyze variant specific glycoprotein (VSG) genes by Southern blotting led to the discovery of the genomic rearrangements involved in antigenic variation (I-5). This chapter will describe the growth of trypanosomes in laboratory rodents. The techniques that this involves start with the growth from a frozen stabilate, which may be a field isolate, and the preparation of further stabilatesfrom infected blood. The basis of the preparation of large numbers of trypanosomes (1 x 109-5 x lOto cells) from blood is the retention of blood cells on a DEAE-cellulose column because of their surface negative charge, while trypanosomes pass through (6,7). These cells then provide the basis for further study, such as the preparation of DNA or RNA (see Chapter 8), the purification of the VSG or ot