John M. S. Bartlett.pdf - Bio-Nica.info

Recombinant PCR 523
3. Because the transformation efficiency is low, highly competent bacteria (transfection
efficiency >1 × 10 9 /µg of monomer pUC19) should be used. Using restriction endonuclease
digested templates, the transformation efficiency is about 10 colonies with the mutation/ng
total DNA used to transform E. coli.
4. After 14 amplification cycles, the PCR product yield is much higher when using a linear
template than when using a supercoiled template.
5. If a plasmid template cannot be linearized outside the region to be amplified before PCR
amplification, the PCR product must be removed from the supercoiled plasmid template.
This can be accomplished either by agarose gel electrophoresis and extraction using
GeneClean or by digestion with the restriction endonuclease DpnI. When agarose gel
resolution and GeneClean extraction are used, the entire PCR product should be gel
purified and reconstituted in 25 to 30 µL of TE, and 2.5 µL is combined with the
2.5 µL of the other PCR product before transformation. If DpnI is used, add 20 U of DpnI
directly to 25 µL of the PCR sample using the recommended 10× DpnI buffer in a final
total volume of 30 µL, and incubate the mixture at 37°C for 1 h. No further purification
of the PCR product is necessary.
6. The exact buffer components and conditions for PCR vary with different primers and
template.
7. There is always the possibility of a sequence error in a single clone after PCR amplification.
The altered region should be sequenced, and one may choose to clone a restriction fragment
containing the mutated or recombined region of interest into a construct that has not
undergone PCR amplification.
Acknowledgments
This work was supported by the Roy J. Carver Charitable Trust, the University of
Iowa through funds generated by the Childrens Miracle Network Telethon, and by
National Institutes of Health grant R01 HG00569. We thank Jim Hartley for suggesting
use of the restriction endonuclease DpnI to remove supercoiled template from the
PCR mixture.
References
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enzymatic amplification of DNA in vitro: the polymerase chain reaction, in Cold Spring
Harbor Symposia on Quantitative Biology, vol. LI, Cold Spring Harbor Laboratory, Cold
Spring Harbor, NY, pp. 263–273.
2. White, B. (1993) Methods in Molecular Biology, vol. 15, PCR Protocols: Current Methods
and Applications, Humana, Totowa, NJ.
3. Jones, D. H. and Howard, B. H. (1991) A rapid method for recombination and site-specific
mutagenesis by placing homologous ends on DNA using polymerase chain reaction.
Biotechniques 10, 62–66.
4. Jones, D. H. (1994) PCR mutagenesis and recombination in vivo. PCR Methods Appl.
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activator of the clcABD chlorocatechol operon in Pseudomonas putida. J. Bacteriol. 175,
417– 427.
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