Comparative evaluation of methods for isolation of plasmids from ruminococci of buffalo rumen origin

Attempts were made to isolate plasmids from 21 Ruminococci isolates by following the methods of Birnboim and Doly [3], Anderson and McKay [1], Klaenhammer [16], Asmundson and Kelly [2], Cocconcelli et al. [8] and Sullivan and Klaenhammer [23]. However, plasmids could be consistently detected from some ruminococcal isolates only by using the method of Sullivan and Klaenhammer [23]. The performance of this method was further improved by incorporationg rumen fluid in the growth medium, pelletting the culture after 8 h or more and overnight freezing and thawing of the cell pellet. By introducing these steps in the protocol, the quality and yield of plasmid DNA were considerably improved. Plasmids were detected in 13 of the ruminococcal isolates. The number of plasmids ranged from one to three and the molecular sizes of the covalently closed circular plasmids ranged from 4.7 to 70 kb. As many as nine isolates harboured only a single plasmid of low molecular weight and only one of the isolates possessed three resident plasmids. Absence of a separate, distinct and sharp plasmid band in cesium chloride - ethidium bromide gradient tubes loaded with the samples were frequently recorded except in case of crude plasmid preparation from OF2 culture. Purified plasmids of reasonable quality could be recovered from the gradient tubes in spite of absence of separate plasmid band though the yields were poor.