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Pfu DNA Polymerase as a Hot Start Alternative

ard deviation) is shown for two independent assays, each performed in duplicate.

All PCR amplifications were performed under identical conditions, using 2.5 U of Taq2000 DNA polymerase, Taq2000 DNA polymerase plus a hot start technique, AmpliTaq Gold DNA polymerase or cloned Pfu DNA polymerase. The following reaction components were added to each Stratagene 600-l thin-wall tube: 250 ng of DNA template, 250 ng of each oligonucleotide primer and 1X of the appropriate reaction buffer in a 100-l ml reaction volume. Taq, cloned Pfu or GeneAmp PCR buffers were used for Taq2000, cloned Pfu or AmpliTaq Gold DNA polymerase amplifications, respectively. Cycling parameters are described in each figure legend. The amplified reaction products were electrophoresed on a vertical 6% acrylamide, 1X TBE gel for 2 hours at 50 V, stained with ethidium bromide and imaged with the Eagle Eye II still video system.

REFERENCES

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

  3. Bergseid, M., et al. (1991) Strategies 4: 34-35.

  4. Kristjansson, J.K. (1992) In Thermophilic Bacteria, p. 7. CRC Press, Boca Raton, Florida.

  5. Lewin, B. (1994) In Genes, Vol V, p.111. Oxford University Press, New York.

  6. Mullis, K.B. (1991) PCR Methods and Applications 1: 1-4.

  7. DAquila, R.T., et al. (1991) Nucleic Acids Res. 19: 3749.

  8. Chou, Q., et al. (1992) Nucleic Acids Res. 20: 1717-1723.

  9. Kellogg, D.E., et al. (1994) Biotechniques 16: 1134-1137.

  10. Dutton, C.M., Payton,
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