![]() This study provides an evaluation of six commonly used methods of cell lysis and DNA extraction of Halococcus spp., and the suitability of the resulting DNA for molecular analysis. For extraction of DNA the lysis buffer will commonly contain SDS. RNase inhibitors are usually present in the lysis buffer, since RNases can be very resistant to denaturation and remain active. Lysis of cells and DNA extraction of samples from spiked sediment proved to be difficult, with the XS-buffer method indicating the best results. What is lysis buffer made of Cell lysis buffer for RNA extraction is highly denaturing and is usually composed of phenol and guanidine isothiocyanate. Using a combination of bead beating, chemical lysis with lysozyme, and thermal shock, lysis of cells was achieved however DNA was badly sheared. Methods based on chemical lysis (Triton X-100, XS-buffer, and CTAB) showed the best results, however, Triton X-100 treatment failed to produce visible DNA fragments. Results showed that boiling and freeze/thawing had little effect on the lysis of both Halococcus strains. The methods tested were based on physical disruption (boiling and freeze/thawing), chemical lysis (Triton X-100, potassium ethyl xanthogenate (XS) buffer and CTAB) and on enzymatic lysis (lysozyme). ![]() ![]() The effects of six different DNA extraction methods were tested on Halococcus hamelinensis, Halococcus saccharolyticus and Halobacterium salinarum NRC-1 as well as on an organic rich, highly carbonated sediment from stromatolites spiked with Halococcus hamelinensis. Furthermore, the lack of a suitable lysis method hinders subsequent molecular analysis. ![]() are known for their rigid cell walls, and are thus difficult to lyse and could potentially be overlooked in an environment. The extraction of nucleic acids from a given environment marks a crucial and essential starting point in any molecular investigation. ![]()
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