Purification and Cloning of pSyma DNA

Sinorhizobium meliloti strain 1021 was grown in TY broth to an OD600=1 (Beringer 1974). Agarose embedded DNA was prepared using a CHEF bacterial genomic DNA plug kit (Biorad) with the modification that more cells were used: approximately 2 x 109 cells for each milliliter of agarose plugs.

Because the circular replicons of S. meliloti are not easily separated, even by pulsed field gel electrophoresis (PFGE; Jumas-Bilak et al. 1998; Sobral et al. 1991), agarose-embedded DNA was digested with the restriction enzyme SwaI (Boeringer Mannheim, 40 units enzyme per plug). SwaI linearizes pSyma into a 1.4 Mb DNA that is easily separated from the chromosomal and pSymb DNAs using a CHEF-DR III apparatus (Biorad) and the following conditions: 1% pulsed field certified agarose (Biorad) in 1/2X TBE buffer, a switch time ramped from 60 s to 120 s over 24 h, 120 degrees field angle and a field strength of 6V/cm. After PFGE, the pSyma band was excised and electroeluted in a dialysis bag, using PFGE and the same conditions described above. The yield of purified pSyma DNA per 14 centimeter gel was approximately 0.3 µg. The pSymA DNA was purified either once or twice by this procedure. DNA from multiple gels was pooled, concentrated, and sheared randomly (Thorstenson et al. 1998). A portion of this DNA, 1 to 2-kb in size, was purified by HPLC, cloned into a linker/adaptor version of M13mp18 in order to minimize chimeras (Hyman et al. manuscript in preparation) and sequenced using the BigDye terminator technology on ABI377-XL sequencers.

Figure 1: PFGE of SwaI S. meliloti DNA

Figure 2: Double purification of pSyma DNA

 

References

Beringer, J.E. 1974. R factor transfer in Rhizobium leguminosarum. J. Gen. Microbiol. 84:188-198.

Jumas-Bilak, E., S. Michaux-Charachon, G. Bourg, M. Ramuz, and A. Allardet-Servent. 1998. Unconventional genomic organization in the alpha subgroup of the Proteobacteria. J. Bacteriol. 180:2749-2755.

Sobral, B.W., R.J. Honeycutt, A.G. Atherly, and M. McClelland. 1991. Electrophoretic separation of the three Rhizobium meliloti replicons. J. Bacteriol. 173:5173-5180.

Thorstenson, Y.R., S.P. Hunicke-Smith, P.J. Oefner, and R.W. Davis. 1998. An automated hydrodynamic process for controlled, unbiased DNA shearing. Genome Res. 8:848-855.

 

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