Potential Sources and Solutions to Genomic DNA Contamination of Plasmid DNA Following Nucleobond AX Purification
When purifying plasmid DNA using the Nucleobond AX alkaline lysis-based protocol, rarely is genomic DNA found as a contaminant in the final eluate from a cartridge. Genomic DNA elutes from a cartridge only if it is added in the first place and, unfortunately, once genomic DNA is bound to a cartridge together with the plasmid there is no simple chromatographic method to separate the two.
(A) There are only a few ways in which genomic DNA can be introduced to a
cartridge. The first of these involves how the cells were handled.
(B) Another source of genomic DNA contamination results from inefficient
pelleting of this DNA with the SDS during the centrifugation that follows the
addition and incubation with buffer S3. There are a number of potential
causes for this as well as a number of possible solutions.
- If the cell suspension was either vortexed or shaken too vigorously after the addition of either buffers S1, S2, or S3, then the bacterial genomic DNA would be sheared and released from the cells. This is the most common mistake made by researchers who are isolating plasmids for the first time. The solution is to be much more gentle during these first few steps of the procedure. Slowly inverting the tube 5-10 times is sufficient for mixing its contents.
- Genomic DNA could also be released if cell lysis was too intensive either because it was done too long or lysozyme was added. If extensive lysis is suspected, then omit the lysozyme (it is not recommended for gram-negative bacteria). Secondly, monitor the time of lysis after the addition of buffer S2 more closely. Finally, incubate the reaction on ice to facilitate the precipitation of the SDS and cellular debris.
Plasmid-Safe ATP-dependent DNAse is the easiest way to salvage critical plasmid preparations in which the genomic contamination is already present. Plasmid-Safe selectively digests linear single- and double-stranded DNA contaminants in plasmid and cosmid preparations. Contact Epicentre Technologies Corporation in Madison, WI at
(800) 284-8474 for more information.
- Perhaps the simplest reason is that at the time of the preparation, there was an insufficient quantity of SDS in buffer S2 to permit complete binding to the cellular debris. This occurs when the buffer is stored at temperatures below 20C, which causes the SDS to precipitate out of solution. If this is the case, incubation of the buffer at 50C for a few minutes will allow the SDS to be completely redissolved.
- A second potential cause for inefficient pelleting of genomic DNA is that the cell suspension was not mixed soon enough after the addition of buffer S3. This could result in the formation of clusters of PDS (potassium dodecyl sulfate) together with cellular debris including genomic DNA. These clusters have a tendency to float. If these floating pieces are added to a cartridge then genomic DNA contamination almost definitely will occur (in addition to a lower cartridge capacity for plasmid DNA). There are a variety of possible solutions to eliminate, or at least minimize, the amount of floating debris after centrifugation.
- The first remedy is to use sterile 6 inch cotton applicators to "fish" the pieces out. This only takes a few seconds per prep and works pretty well.
- The second alternative is to do the centrifugation of the prep at a higher speed and for a longer period of time. For example, instead of 38 minutes at 12000 x g for an AX-100, it can be done for 45 minutes at 15000 rpm and 4C. A second centrifugation can also be performed on the supernatant prior to its addition to a cartridge.
- The third alternative is to add the neutral detergent Triton X 100 to a concentration of 0.02% just after the S2 buffer is added. The manufacturer, Macherey-Nagel, has performed experiments that showed that this addition resulted in the formation of harder pellets during the centrifugation which, therefore, reduced the amount of floating material. The reason that Triton X is not already included in buffer S3 is that it is a potent inhibitor of many enzyme reactions and should be used only as a last resort.
Having specific problems? Please refer to the Nucleobond AX Trouble Shooting Guide.
Back to the Nucleobond AX applications guide