( of 5 mM Tris-HCl pH 8.0 and...)Identification of Differentially Expressed Gene Products with the CastAway,,,System*

of 5 mM Tris-HCl, pH 8.0 and 0.1 mM EDTA and was incubated at 65C for 2 hours. The pipet tip was removed, and the tube was centrifuged for 5 to 10 seconds in a microcentrifuge. Supernatants from the eluted gel slices were used as templates in PCR amplifications using the original RAP-PCR primers. The reamplified RAP-PCR products were analyzed by agarose gel electrophoresis figure 3. Products of appropriate size were obtained with all 14 samples. To verify the efficacy of the fragment isolation by aspiration, the gel plate was dried and exposed to film for a third time (data not shown). Silver staining was missing from the film at positions corresponding to the eluted bands, confirming that aspiration had effectively removed the band of interest from the hydrated gel.

figure 3

Conclusions

Techniques such as RAP-PCR and DD have wide application because of their ability to detect global changes in gene expression. However, both approaches require substantial time and effort for analyzing the identified gene products. Stratagenes new CastAway precast sequencing system can be used to expedite the process by reducing the time and effort necessary to detect and isolate differentially expressed gene products.

REFERENCES

1. McClelland, M., and Welch, J. (1994) PCR Methods Appl. 4: S66-S80.

2. Liang, P., Bauer, D., Averboukh, L., et al. (1995) Methods Enzymol. 254: 304-321.

3. Zhao, S., Ooi, S., and Pardee, A. (1995) Biotechniques 18: 842-850.

4. Zhang, H., Zhang, R., and Liang, P. (1996) Nucleic Acids Res. 24: 2454-2455.

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