Plasmid Preparation from Gram-Positive Bacteria Using Nucleobond AX Cartridges

The following procedure is adapted from an article in the December 1993 issue of BioTechniques (Assaf, N. A. and W. A. Dick. 1993. Spheroplast Formation and Plasmid Isolation from Rhodococcus sp. BioTechniques 15:1010-1015.). This paper presents one possible way of bolstering plasmid yields from gram-positive bacteria through a more complete digestion of their cell walls. The procedure below varies from that in the paper in that Nucleobond AX is used in place of CsCl banding to complete the final purification, thus making it more rapid and cost efficient.

In the Assaf and Dick study, Rhodococcus were grown in 200 ml cultures at 28C on a rotary shaker until the OD reading at 600 nm reached 0.5 before starting the preparation.

1. Pellet the cells by centrifugation at 2000 x g for 5 minutes. Wash the cells with 0.2 culture volumes of 5 mM EDTA, pH 6.8. Wash the cells a second time with 0.2 culture volumes of 20 mM Tris-HCl, pH 6.8. Resuspend the cells in 20 mM Tris-HCl, 1 mM MgCl2, pH 6.8 to an OD600 reading of 0.5 (approximately in the same volume as the original culture).
2. Add mutanolysin to a final concentration of 5 U/ml and incubate at 37C for 1 hour.
3. Pellet the cell by centrifugation at 2000 x g for 5 minutes. Resuspend the cells in the appropriate volume of buffer S1 for the particular Nucleobond AX cartridge to be used following the low copy number plasmid rule above, that is, use a minimum of 4.0 ml of S1 buffer for each 100 ml of culture or the recommended volume of S1 for the cartridge being used, whichever volume is greater.
4. Add lysozyme to a final concentration of 25 mg/ml and incubate at 37C for 30 minutes.
5. Begin at step 2 in the Procedure for the Purification of Plasmids and Cosmids when buffer S2 is added and follow the procedure to completion, again while adhering to the low copy number plasmid rule above. To reiterate, use a minimum of 4.0 ml of each of the three lysis buffers for each 100 ml of culture or the recommended volumes of lysis buffers for the cartridge being used, whichever volumes are greater.

Having specific problems? Please refer to the Nucleobond AX Trouble Shooting Guide.

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