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prostar RT-PCR Systems for Robust High-Fidelity RNA Amplification

0 minutes (targets >2 kb). PCR amplification reactions (50 l) contained 1X Ultra-HF PCR buffer, 200 M each dNTP, forward and reverse gene-specific primers (2 ng/l of each), 1 l of cDNA, and 2.5 U of PfuTurbo DNA polymerase. PCR amplification was performed as described above, except that extension at 68C was carried out for 3 minutes/kb.

RT-PCR products were electrophoresed on 0.8 to 1% agarose/1X TBE gels, stained with ethidium bromide, and imaged using the Eagle Eye II still video system. Lanes labeled M contain the Lambda/Hind III-fX174/Hae III DNA markers.

REFERENCES

  1. Powell, L.M. et al. (1987) Cell 50: 831-840.

  2. Kawasaki, E.S., et al. (1988) Proc. Natl. Acad. Sci. 85: 5698-5702.

  3. Frohman, M.A. et al. (1988) Proc. Natl. Acad. Sci. 85: 8998-9002.

  4. Myers, T.W. and Gelfand, D.H. (1991) Biochemistry 30: 7661-7666.

  5. Tellier, R., et al. (1996) Proc. Natl. Acad. Sci. 93: 4370-3.

  6. Roberts, J.D., Bebenek, K., and Kunkel, T.A. (1988) Science 242: 1171-3.

  7. Cline, J., Braman, J.C., and Hogrefe, H.H. (1996) Nucl. Acids Res. 24: 3546-3551.

  8. Hogrefe, H., Bai, F., and Cline, J. (1998) Strategies 11: 36-37.

  9. Hogrefe, H., et al. (1997) Strategies 10: 93-96.

  10. Nielson, K.B,. et al. (1997) Strategies 10: 29-32.

  11. Guide to Pfu DNA Polymerase. (1996) Stratagene, La Jolla, California

* U.S. Patent No. 5,545,552 and patents pending
** U.S. Patent No. 5,556,772 and patents pending


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