Basically there are only four ways in which a seemingly insoluble pellet can occur following precipitation of the eluate from a Nucleobond AX cartridge. These are (1) the pellet was over dried or under dried, (2) the precipitation was centrifuged too long, (3) there is resin in the precipitate, and (4) KCl was also precipitated. All except item 3 are not product related and, in those cases, the preps are usually not salvageable. However, they can all be easily avoided in the future. Each will be discussed in turn.
(1) Nucleic acid pellets become over dried through centrifugation in a vacuum making them very difficult to dissolve. Many researchers tend to dry pellets in this manner, but unfortunately they tend to do it too long. This is further complicated by the fact that different pellets have different levels of residual moisture. The best advice to give is to not vacuum dry. Centrifuge tubes should be drained of excess fluids by inverting and resting on paper towels or Kim Wipes and then carefully swabbed with sterile, 6 inch, cotton tip applicators. When over drying occurs, some of the prep may be salvaged by dissolving the pellet in TE or water at about 60C with periodic vortexing (not recommended for genomic DNA preps where large fragment size is critical).
Pellets can be under dried also and the alcohol present in these cases can result in their insolubility. (Even if the pellet dissolves, trace amounts of alcohol can come back to haunt you in future manipulations. Anyone who has seen a sample mysteriously disappear from a well on an agarose gel can attest to this problem.) Again, thoroughly draining and swabbing of centrifuge tubes should alleviate this problem.
(2) Nucleic acids can be over centrifuged so as to form an insoluble mass. Typically, it is a combination of time and g force. It is usually a problem with smaller preps where a microfuge is being used. In this case 5-10 minutes at top speed is sufficient. For larger preps in which a fixed angle rotor is employed, 10-15 minutes at 12,000 x g and 4C is sufficient. When over centrifugation occurs, some of the prep may be salvaged by heating and periodic vortexing, as in (1) above.
(3) Resin usually winds up in the prep when force is applied to the cartridge during the elution step. In older kits users were instructed to "push out" the cartridge for maximum recovery. This is not necessary and the same result can be achieved by the addition to the cartridge of one more column volume of the elution buffer. When it occurs, this problem is easily resolved. The prep should be centrifuged in a microfuge for 1-2 minutes to pellet all of the resin and the DNA-containing supernatant should be carefully removed. (This may have to be repeated 2-3 times to ensure the complete removal of the resin and may tend to lower yields, since at low pH and low ionic strength the resin will bind the DNA. This binding will not occur if the prep is in the N5 buffer.)
(4) Salt pellets typically are the result of either (a) using an alcohol other than iso-propanol for the precipitation (usually ethanol) or (b) using iso-propanol that has not been equilibrated to room temperature, that is, it is too cold.
Occasionally salt pellets do occur for reasons other than these. If a particular lab repeatedly experiences this problem for no apparent reason then they can dilute the eluant 1:1 in water or TE prior to the addition of the iso-propanol. This will have essentially no effect on how well the nucleic acids precipitate.
In order to salvage as much as possible when a salt pellet occurs, dissolve the pellet in water, add 0.7 volumes of iso-propanol to re-precipitate, and centrifuge again. Typically no additional salt is needed to re-pellet the nucleic acid, but in some cases the addition of sodium acetate to a final concentration of 0.3M may be required.
Having specific problems? Please refer to the Nucleobond AX Trouble Shooting Guide.
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