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. 1991 Jan 25;19(2):385–390. doi: 10.1093/nar/19.2.385

An automated method for DNA preparation from thousands of YAC clones.

A J MacMurray 1, A Weaver 1, H S Shin 1, E S Lander 1
PMCID: PMC333606  PMID: 2014175

Abstract

We describe an automated method for the preparation of yeast genomic DNA capable of preparing thousands of DNAs in parallel from a YAC library. Briefly, the protocol involves four steps: (1) Yeast clones are grown in the wells of 96-well microtiter plates with filter (rather than plastic) well-bottoms, which are embedded in solid growth media; (2) These yeast cultures are resuspended and their concentrations determined by optical density measurement; (3) Equal numbers of cells from each well are embedded in low-melting temperature agarose blocks in fresh 96-well plates, again with filter bottoms; and (4) DNA is prepared in the agarose blocks by a protocol similar to that used for preparing DNA for pulsed-field gels, with the reagents being dialyzed through the (filter) bottoms of the microtiter plate. The DNA produced by this method is suitable for pulsed-field gel electrophoresis, for restriction enzyme digestion, and for the polymerase chain reaction (PCR). Using this protocol, we produced 3000 YAC strain DNAs in three weeks. This automated procedure should be extremely useful in many genomic mapping projects.

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Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Burke D. T., Carle G. F., Olson M. V. Cloning of large segments of exogenous DNA into yeast by means of artificial chromosome vectors. Science. 1987 May 15;236(4803):806–812. doi: 10.1126/science.3033825. [DOI] [PubMed] [Google Scholar]
  2. Carle G. F., Olson M. V. An electrophoretic karyotype for yeast. Proc Natl Acad Sci U S A. 1985 Jun;82(11):3756–3760. doi: 10.1073/pnas.82.11.3756. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Chu G., Vollrath D., Davis R. W. Separation of large DNA molecules by contour-clamped homogeneous electric fields. Science. 1986 Dec 19;234(4783):1582–1585. doi: 10.1126/science.3538420. [DOI] [PubMed] [Google Scholar]
  4. Church G. M., Gilbert W. Genomic sequencing. Proc Natl Acad Sci U S A. 1984 Apr;81(7):1991–1995. doi: 10.1073/pnas.81.7.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Feinberg A. P., Vogelstein B. A technique for radiolabeling DNA restriction endonuclease fragments to high specific activity. Anal Biochem. 1983 Jul 1;132(1):6–13. doi: 10.1016/0003-2697(83)90418-9. [DOI] [PubMed] [Google Scholar]
  6. Fogel S., Welch J. W. Tandem gene amplification mediates copper resistance in yeast. Proc Natl Acad Sci U S A. 1982 Sep;79(17):5342–5346. doi: 10.1073/pnas.79.17.5342. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Mullis K. B., Faloona F. A. Specific synthesis of DNA in vitro via a polymerase-catalyzed chain reaction. Methods Enzymol. 1987;155:335–350. doi: 10.1016/0076-6879(87)55023-6. [DOI] [PubMed] [Google Scholar]
  8. Reed K. C., Mann D. A. Rapid transfer of DNA from agarose gels to nylon membranes. Nucleic Acids Res. 1985 Oct 25;13(20):7207–7221. doi: 10.1093/nar/13.20.7207. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Saiki R. K., Gelfand D. H., Stoffel S., Scharf S. J., Higuchi R., Horn G. T., Mullis K. B., Erlich H. A. Primer-directed enzymatic amplification of DNA with a thermostable DNA polymerase. Science. 1988 Jan 29;239(4839):487–491. doi: 10.1126/science.2448875. [DOI] [PubMed] [Google Scholar]
  10. Shin H. S., Flaherty L., Artzt K., Bennett D., Ravetch J. Inversion in the H-2 complex of t-haplotypes in mice. Nature. 1983 Nov 24;306(5941):380–383. doi: 10.1038/306380a0. [DOI] [PubMed] [Google Scholar]

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