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Simple Approaches for Optimization of RT-PCR

he amount of RT-PCR enzyme mix in the reaction can also dramatically increase the yield of product (Fig. 5).

Conclusion

Simple factors in RT-PCR, such as primer concentration, primer annealing temperature, and enzyme amount, have profound effects on sensitivity, specificity, and yield. Optimization of these factors is an excellent starting point for achieving desirable RT-PCR results.

References

1. GOBLET, C., PROST, E., AND WHALEN, R.G. (1989) Nucleic Acids Res. 17, pp. 2144.

2. SAMBROOK, J. AND RUSSEL, D.W. (2001) Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, pp. 8.46-8.53.

3. SELLNER, L.N., COELEN, R.J., AND MACKENZIE, J.S. (1992) Nucleic Acids Res. 20, pp. 1487- 1490.

4. ROTH, M.J., TANESE, N., AND GOFF, S.P. (1985) J. Biol. Chem. 260, pp. 9326-9335.

5. SAIKI, R.K., GELFAND, D.H., STOFFEL, S., SCHARF, S.J., HIGUCHI, R., HORN, G.T., MULLIS, K.B., AND ERLICH, H.A. (1988) Science 239, pp. 487-491.

6. SAMBROOK, J. AND RUSSEL, D. W. (2001) Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, pp. 8.13-8.16.

7. http://www-genome.wi.mit.edu/cgi-bin/primer/primer3_www.cgi

8. PUISSANT, C. AND HOUDEBINE, L.-M. (1990) BioTechniques 8, pp. 148-149.

9. SAMBROOK, J. AND RUSSEL, D. W. (2001) Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, pp. 7.9-7.12.

10. SAMBROOK, J. AND RUSSEL, D. W. (2001) Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, pp. 7.82-7.84.


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