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 means of computerized series research, the current quantity bridges the distance among the protocols provided with software and response kits and the finalized facts awarded in th. Read more...
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Extra info for Automated DNA Sequencing and Analysis
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.