The Apoptotic DNA Ladder Kit from Roche Molecular Biochemicals is designed to isolate DNA from starting material so that the typical DNA ladder produced by apoptotic cells can be visualized on an agarose gel. The kit, which can be used with either whole blood or cultured cells, contains buffers for lysis of the starting material, washing and elution of bound DNA, filter and collection tubes, and a positive control consisting of lyophilized apoptotic cells.
The process is fairly straightforward: starting material is lysed in binding buffer, then applied to microfuge tubes containing glass fiber fleece, which binds nucleic acids. After a brief incubation, the tubes are washed and eluted with elution buffer. The eluted samples are then run on an agarose gel and stained to visualize the DNA ladder.
Before buying this kit, I tried repeatedly to isolate DNA from cultured cells and visualize the ladder using standard protocols, which include time-consuming RNAse and proteinase K treatments to reduce viscosity. However, the samples remained highly viscous, resulting in streaky gels that were difficult to interpret. The Apoptotic DNA Ladder Kit solved the problem of viscosity and reduced the performance time to about 35 minutes.
The only drawbacks to this method are inherent to apoptosis studies rather than problems with the Apoptotic DNA ladder kit. The ability to detect the ladder depends on optimizing the treatment conditions so that a high enough percentage of the cells are undergoing apoptosis. Also, some cell types do not produce a typical DNA ladder when undergoing apoptosis. Either of these problems can result in a long DNA smear instead of a clearly visible ladder. The positive control is very useful in these cases, because it helps you determine whether your samples contain an appropriate amount of DNA. Also, if using volumes or amounts of starting material other than the recommended 2 x106 cells/200 microliters, it may take some time to work out an appropriate sample volume that can be easily visualized on a gel.
The Apoptotic DNA Ladder Kit from Roche Molecular Biochemicals is fast and easy to use, and produces cleaner DNA samples than standard protocols in a shorter period of time.
Kimi Nishikawa
Graduate Student
State University of New York at Albany