By Priya Handa, Swapna Thanedar, Umesh Varshney (auth.), Jeff Braman (eds.)
Hands-on researchers with confirmed tune documents describe in stepwise model their complicated mutagenesis recommendations. The members specialise in advancements to standard site-directed mutagenesis, together with a bankruptcy on chemical site-directed mutagenesis, PCR-based mutagenesis and the variations that permit excessive throughput mutagenesis experiments, and mutagenesis according to gene disruption (both in vitro- and in situ-based). extra tools are supplied for in vitro gene evolution; for gene disruption according to recombination, transposon, and casette mutagenesis; and for facilitating the advent of a number of mutations. Time-tested and hugely sensible, the protocols in In Vitro Mutagenesis Protocols, 2d variation provide ultra-modern molecular biologists trustworthy and robust concepts with which to light up the proteome.
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23, 4530–4531. 17. Gorelov, V. , Roher, H. , and Goretzki, P. E. (1994) A method to increase the sensitivity of mutation specific oligonucleotide hybridization using asymmetric polymerase-chain reaction (PCR). Biochem Biophys Res Commun. 200, 365–369. 18. Wooddell, C. I. and Burgess, R. R. (1996) Use of asymmetric PCR to generate long primers and single-stranded DNA for incorporating cross-linking analogs into specific sites in a DNA probe. Genome Res. 6, 886–892. 19. Millican, D. S. and Bird, I.
John Wiley & Sons, New York, NY, p. 1 12. , and Truyen, U. (1995) A simple touch-down polymerase chain reaction for the detection of canine parvovirus and feline panleukopenia virus in feces. J. Virol. Methods 55, 427–433. 13. , and Jin, L. (1998) Fluorescence-based semi-automated gene scan with microsatellite markers by multiplex PCR techniques. Chung Hua I Hsueh I Chuan Hsueh Tsa Chih 15, 104–107. Two-Stage PCR Using QuikChange SDM 43 14. Kim, Y. G. and Maas, S. (2000) Multiple site mutagenesis with high targeting efficiency in one cloning step.
3. Stop the reaction by heating at 65°C for 10 min. 4. 0 µL phosphorylated primer solution in each mutagenesis reaction. 4. Mutagenic Strand Extension 1. 5-mL tube (see Note 10). Adjust with H2O to a total volume of 20 µL. Mix well, and briefly centrifuge. 2. Incubate at 100°C for 3 min to completely denature the plasmid (see Note 11). 3. Chill immediately in an ice water bath (0°C) for 5 min (see Note 12). 4. To the primer/plasmid annealing reaction, add 3 µL of 10X synthesis buffer, 1 µL T4 DNA polymerase (3 U), 1 µL T4 DNA ligase (5 U), and 5 µL ddH2O (final volume 30 µL).