Improved isolation of genomic DNA from mycobacteria in agarose plugs by rapid lysis with a combination of N-acetylglucosaminidase and lysozyme.

tinely isolate 80-100 yg of RNA per 8 mL of culture. RNA isolated by this method or with TRIzol (manufacturer's recommended procedure) exhibited an A260-280 ratio of 1.6-1.8. When needed, RNA samples were supplemented with RNA loading dye [0.72 mL formamide, 0.16 mL lox MOPS buffer, 0.26 mL 37% formaldehyde, 0.18 mL distilled water (DEPC-treated), 0.10 mL 80% glycerol, and 8.0 mg bromophenol blue] and resolved on 1.2% formaldehyde-agarose gels. Alternatively, bromophenol blue (50 mg per 100 mL) could be added to the lysis buffer, and RNA, isolated as described above, could be directly applied to formaldehyde-agarose gels. RNA isolated from P. gingivalis and E. coli using this new method was fractionated on formaldehyde-agarose gels along with preparations obtained by the TRIzol method. Figure 1 compares the quality of RNA isolated by this new protocol relative to the TIUzol method. No differences are seen in Figure 1, indicating no visible contamination in the new procedure. In the TRIzol method, chromosomal DNA contamination was detected for both organisms and was removed by DNase (RNase free) digestion prior to electrophoresis. RNA samples resolved on a formaldehyde-agarose gel were then transferred to a HybondTM-N+ nitrocellulose membrane (Amersham, Arlington Heights, IL, USA) by standard methods (3). This Northern blot was subjected to hybridization with a 0.4-kb PstI-BamHI DNA fragment internal to the F? gingivalis hemR gene (Karunakaran and Kuramitsu, unpublished results) as a probe utilizing nonradioactive ECLTM direct nucleic acid labeling and detection systems (Amersham). Figure 2 shows the results obtained following exposure of the blot to HyperfilmTM-ECL (Amersham). The results clearly indicate a transcript size of 3.1 kb for the F? gingivalis hemR gene with RNA isolated by both methods. These results indicate that the newly described method represents a rapid and simple method for isolating highquality RNA from gram-negative bacteria. Northern blot analysis readily detects specific mRNA transcripts in these preparations.