When performing plasmid preparations it is always desirable to maximize the yield of DNA. The fewer preparations that have to be done, the more time there is to pursue more fruitful experiments. High copy number plasmids are useful in this case and they can be isolated in milligram quantities from cultures as small as 200 ml. This being the case, it is important to choose the correct size of Nucleobond AX cartridge for one's plasmid isolation needs. If multi-milligram quantities are expected, then the AX-2000 or the AX-10000 cartridges should be used. However, if only smaller cartridges are available or if more then 10 mg are anticipated and the AX-10000 is being used, then the following procedure may be useful in order to extract as much plasmid as has been produced. (NOTE 2)
(1)Follow the Nucleobond purification procedure for the lysis of bacteria and the removal of the cellular debris (buffers S1, S2, and S3, and so on).
(2)Apply the supernatant to the cartridge as described, collect the entire flowthrough from the cartridge, and place the flowthrough on ice.
(3)Continue with the purification procedure, that is, add to the cartridge the recommend N3 washing buffer volume and the recommended N5 elution buffer volume and collect plasmid.
(4)Immediately flush the column with deionized water by filling it to the top rim and allowing it to completely run out. Re-equilibrate the cartridge with the prescribed amount of buffer N2, and reapply the recovered flowthrough from step (2) above. Wash and elute as usual. (NOTE 3)
(5)Repeat step (4) until the flowthrough has been depleted of plasmid. (This can be determined by checking the UV absorbance at 260 nm of the various eluates.)
(6)Combine the plasmid-containing eluates and precipitate (or precipitate the various eluates separately, if there are volume constraints).
NOTE 1: In order to avoid cross contamination, cartridge reuse is only recommended for purification of the same nucleic acid. For example, when purifying plasmids, a cartridge to be reused should be dedicated to a single plasmid.
NOTE 2: Use of this procedure may deplete the N series of buffer solutions supplied with the Nucleobond kits. Please refer to N series buffer preparation.
NOTE 3: For longer term storage, flush the column with a 50% ethanol solution after the wash with deionized water.Wrap the cartridge in plastic wrap and store at 4C until needed.
Having specific problems? Please refer to the Nucleobond AX Trouble Shooting Guide.
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Updated 1/20/98