Automated DNA Sequencing and Analysis by Mark D. Adams, Chris Fields, J. Craig Venter

By Mark D. Adams, Chris Fields, J. Craig Venter

A well timed ebook for DNA researchers, computerized DNA Sequencing and research reports and assesses the state-of-the-art of computerized DNA series analysis-from the development of clone libraries to the developmentof laboratory and neighborhood databases. It provides the methodologies and methods of automatic DNA series research in a manner that enables them to be in comparison and contrasted. through taking a large view of the Read more...

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2. PCR includes o nly three major ste ps, technically similar to the first thr ee steps in the M13 protocol. It elimina tes vector DNA and also pro vides a 5- to 10-fold higher DNA concentration, but it is mor e expe nsive and has many limits on process scaling. On e solutio n for a high PCR throughput is the BioOven (BioThe rm, Fairfax , VA ), which can acco mmodate up to six plates. 5 M NaCI. Furth er proce ssing of the filters is the same as in standard South ern blot procedures. Produ ction-size filters comprising two-, four -, or six-plate patt ern s and 27 648 to 82 944 dots can be prep ared .

1991) Methods: Companion Methods Enz ymo l. 40. , Kelley. , Tra pp. S.. L. , Fields, e. e. (1992) Nature Genet. 1, 348-353. Sarnbrook, J .. F. & Maniatis, T. (1989) Molecular Cloning: A Laboratory Manual , 2nd edn. Cold Spring Harbor Laboratory Press, Cold Spring Harbor , NY. E. & Berg, e. M. (1990) Proc. Natl. Ac ad. Sci. A . 87, 6213-6217. CHAPTER SIX Shotgun Cloning as the Strategy of Choice to Generate Templates for High-throughput Dideoxynucleotide Sequencing A. BODENTEICH, S. -F. A. ROE Department of Chemistry and Biochemistry.

5 and 3 kb have bound to the membran e . 1 mM E DT A , 20 mM Tri s, pH 8) to eliminate residual agarose. 5 vol. of etha nol. 3 M sodium ace ta te is performed to remov e a ny NaCI residu e . T he pelleted DN A is resuspended in water , and an aliquot is analyzed by agaro se gel electropho resis to determine th e DNA concentration. 8% agaro se gel made fro m low-melting-point aga r and T ris-aceta te bu ffer ( IX T A E ). After electrophoresis, th e bands in the desired size ran ge a re excised from th e gel and melt ed by heating at 6SOC for 10 min.

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