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(MATer, ura3 trpl ade2-101 canl-lO0 leu2-3, 112 his3-6). The quality of the library was monitored during construction in two ways. Initially, the presence of rat DNA in the transformants was assessed by a rapid PCR assay to detect the rat B2 repeat element. This element is highly homologous to the murine B2 (denDunnen and Schoenmakers 1987). The assay and primers used are described by Kusumi and associates (1993) and Haldi and colleagues (1996). Five percent of the transformants from each ligation were screened, and typically greater than 95% of the clones yielded a PCR product visible by agarose gel electrophoresis with ethidium bromide. The insert sizes of the YACs were determined by pulsed field gel electrophoresis as described by Haldi and coworkers (1996). Approximately 1.5% of the YACs from each ligation were examined. Figure 1 shows the distribution of YAC sizes in the 631 clones tested. The mean size is 820 kb, and the median size in 815 kb. Based on the estimated length of the rat genome as 3 billion bp, this library provides 11-fold coverage. Were there not systematic cloning biases in YAC libraries, this would correspond to a probability of 99.995% of a given locus being represented in the library. Thus, any gaps in coverage are likely to be due to cloning biases. Our experiences with human YAC libraries (Hudson et al. 1995) suggest the actual coverage is likely to be 98% of the gehome. |
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