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Comparing Fidelity and Performance of Proofreading PCR Enzymes

follows: 1 cycle at 95C for 1 minute, followed by 30 cycles at 95C for 1 minute (denaturation); 58 to 65C for 1 minute (annealing); and 72C for 1 minute per kb of target; and 1 final extension cycle of 72C for 10 minutes. Identical cycling conditions were used for Pfx, except that a 68C extension temperature was used as recommended by the manufacturer. PCR products were electrophoresed on 1% agarose/1XTBE gels, stained with ethidium bromide and imaged using the Eagle Eye II still video system. Lanes labeled M contained the kb-DNA markers.

REFERENCES

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  2. Flaman, J-.M. Frebourg, et al. (1994) Nucleic Acids Res. 22: 3259-3260.

  3. Andre, P., et al. (1997) Genome Res. 7: 843-852.

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

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

  6. Tagaki, M., et al. (1997) Appl. Environ. Microbiol. 63: 4504-4510.

  7. Perler, F.B., Kumar, H., and Kong, H. (96) Adv. Protein Chem. 48: 377-435.

  8. Westfall, B., et al. (1999) Focus 21: 46.

  9. Guide to Pfu DNA Polymerase, Stratagene, 1996.

  10. Lundberg, K.S., et al. (1991) Gene 180: 1-6.

  11. Nielson, K.B., Cline, J. and Hogrefe, H. (1997) Strategies 10: 40-43.

* U.S. Patent Nos. 5,545,552, 5,866,395, and 5,948,663 and patents pending
** Patents pending


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