Potential Sources and Solutions to Relaxed and Linear Forms in Nucleobond AX Plasmid DNA Preparations

When isolating plasmid DNA using the Nucleobond AX alkaline lysis-based protocol, occasionally relaxed circular and, much more infrequently, linear forms of the plasmid are found as a contaminant in the final eluant from a cartridge. These forms of plasmid DNA can only be eluted from a cartridge if they are added in the first place and, unfortunately, once these DNAs are bound to a cartridge together with the supercoiled form there is no simple method to separate them. There are only a few ways in which relaxed circular and linear plasmids can be introduced to a cartridge. Each will be discussed in turn.

(A) Supercoiled plasmids can be mechanically sheared into the less desirable forms. This occurs when the cell suspension from which they were isolated was either vortexed or shaken too vigorously after the addition of either buffers S2 or S3. The basic rule of thumb is that the larger the plasmid, the more susceptible it is to such shearing. (Handling that is too vigorous would also result in bacterial genomic DNA shearing and release from the cells. Such a prep would be a real mess!!) The solution to this problem is handle the prep more gently during these first few steps of the procedure. Slowly inverting the tube 5-10 times is sufficient for mixing its contents.

(B) Another source of non-supercoiled plasmid contamination involves the strain of bacteria from which the plasmid was purified. Some strains of E. coli (for example, HB101) are endA+ and thus produce the enzyme, endonuclease A. If this enzyme is not quickly and completely removed, it could potentially nick, linearize, and, eventually, completely degrade supercoiled plasmids. (The enzyme is Mg++-dependent so that the problem might not be apparent at the end of a prep.) One solution to this problem is to transfer your plasmids to an endA strain. If this is not possible, then the alternative is to perform the purification procedure without delay, at least until after the cartridge washes have been done. That is to say, don't exceed the recommended times for lysis and neutralization (this could cause other problems anyway) and perform the cartridge washes immediately after the sample has been loaded. This will minimize the exposure of the plasmids to the enzyme, which will be removed during the wash steps.

(C) Relaxed, circular plasmids can occur if the bacterial culture was harvested prematurely. That is, if the alkaline lysis is performed on a culture that is in the lag or log phase of growth then a larger percentage of the purified plasmid will be relaxed (not necessarily nicked). This is a result of the way in which plasmids replicate, each time forming one supercoiled and one nicked, relaxed circle that is quickly closed, but not supercoiled until later. In the log phase, cells are still dividing and thus plasmids are still replicating. The simplest solution here is to grow the culture overnight (12-16 hours) to the stationary phase or saturation before starting.

(D) Finally, nicked, circular plasmids can be the result of something known as a relaxation complex. This DNA-protein complex involves supercoiled plasmid and a set of three tightly bound proteins, one of which is a nuclease. Under certain conditions, which includes treatment with alkali and detergents, the nuclease nicks one strand thereby relaxing the supercoil. Additionally one of the proteins becomes covalently bound to the 5'-P terminus of the nick. As in step (B), the solution to this problem is to perform the purification procedure without delay, at least until after the cartridge washes have been done. If the problem persists, incubate the prep on ice instead of room temperature after the addition of buffer S2. This is also the most critical step as far as time is concerned. The incubation should not exceed five minutes.

In most of the cases above, with the possible exception of (B), the supercoiled plasmid that is present in the prep can be purified away from the other forms by following the Re-Melt and Filter Procedure, which describes a quick procedure for recovering DNA from low-melting-point agarose gel slices using Nucleobond AX.

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

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Updated 1/20/98