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

Unknown Genomic Sequences 383
5. The annealing temperature and the optimal magnesium concentration may be optimized
specifically for each primer, but the given conditions worked reasonably well for most
of the primers we tested.
6. In general, beads are concentrated by placing the 1.5-mL reaction tube on the magnetic
rack for 10 to 20 s. If left for too long, the bead pellets become too tight and therefore
difficult to resuspend. The supernatant is removed with a pipet tip with the tube still in
the rack. Great care is taken at every step to resuspend the beads well by pipetting the
suspension up and down. A drying of the bead pellets should also be avoided.
7. The reagents used for sequencing (see Subheadings 2.8. and 3.8.) are available as a kit
(Circumvent sequencing kit, NE Biolabs).
8. Subheading 3.8., step 19 allows the separation of the labeled fragments from the beads,
which facilitates gel loading. However, the presence of the beads in the sample load does
not adversely affect the migration of DNA fragments.
References
1. Sanger, F., Nicklen, S., and Coulson, A. R. (1977) DNA sequencing with chain-termination
inhibitors. Proc. Natl. Acad. Sci. USA 74, 5463–5467.
2. Pfeifer, G. P., Steigerwald, S. D., Mueller, P. R., Wold, B., and Riggs, A. (1989) Genomic
sequencing and methylation analysis by ligation mediated PCR. Science 246, 810–813.
3. Mueller, P. R., Garrity, P. A., and Wold, B. (1993) Ligation mediated PCR for genomic
sequencing and footprinting, in Current Protocols in Molecular Biology vol. 2 (Ausubel,
F. M., Brent, R., Kingston, R. E., Moore, D. D., Seidman, J. G., Smith, J. A., et al, eds.),
Current Protocols, New York, pp. 15.5.1–15.5.26.
4. Quivy, J.-P. and Becker, P. B. (1994) Direct dideoxy sequencing of genomic DNA by
ligation-mediated PCR. Biotechniques 16, 238–241.
5. Quivy, J.-P. and Becker, P. B. (1993) An improved protocol for genomic sequencing and
footprinting by ligation-mediated PCR. Nucleic Acids Res. 21, 2779–2781.
6. Sambrook, J., Fritsch, E. F., and Maniatis, T. (1989) Molecular Cloning: A Laboratory
Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY.
7. Wu, C. (1989) Analysis of hypersensitive sites in chromatin. Methods Enzymol. 170,
269–289.
8. Bellard, M., Dretzen, G., Giangrande, A., and Ramain, P. (1989) Nuclease digestion of
transcriptionally active chromatin. Methods Enzymol. 170, 317–346.