DNA Sequencing Protocols (Methods in Molecular Biology Vol by Hugh G. Griffin

By Hugh G. Griffin

Institute of foodstuff examine, Norwich, U.K. tools in Molecular Biology, quantity 23. handbook of certain sensible techniques for quite a lot of DNA sequencing equipment. Plastic spiral binding. comprises template training, sequencing reactions, gel pouring, and extra. forty two members, thirteen U.S. DNLM: series research, DNA.

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The central requirements for success are the presence of multivalent cations, an incubation temperature close to O”C, and a carefully controlled heatshock at 42OC,but a number of other compounds and procedures have been found to increase efficiency in some or all strains of E. coli that have been tested. The mechanisms involved in DNA transformation of cells are not fully understood and even with the most efficient methods that are available the proportion of cells that become “competent” for transformation is limited to around 10% of the total population.

125-169. 2. , Bankier, A. , Deininger, P. , Farrell, P. , Gibson, T. , Hatfull, G , Hudson, G. , Satchwell, S. C , Segum, C , Tuffnell, P S , and Barrell, B. G. (1984) DNA sequence and expression of the B95-8 EpsteinBarr virus genome. Nature 310,207-211 3. Davison, A. J. and Scott, J E (1986) The complete DNA sequence of vancellazoster virus J. Gen. Virol 67, 1759-l 816 4. Messing, J. and Bankier, A T (1989) The use of single-stranded DNA phage in DNA sequencing, in Nucleic Acids Sequencing. A Practical Approach (Howe, C.

Gene Anal Tech. FTER 9 Sequential Deletions of Single-Stranded DNA “Cyclone Sequencing” George Murphy 1. Introduction A major problem of the directed deletion of double-stranded DNA using Exonuclease III (Chapter 8) is the necessity to find two enzyme sites to open up the insert for digestion and to protect the primer site. The “Cyclone Sequencing” technique of Dale, McClare, and Houchins (1) overcomes this problem as it is totally independent of the restriction sites in the insert. In this procedure single-stranded Ml3 is hybridized to an oligomer that spans the Hind111(even-numbered M 13 phage) or EcoRI site (odd-numbered phage) of the polylinker, generating a region of double-stranded DNA.

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