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Nonfiction 3

By ICM-1920, Strasbourg.

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Southern Blotting and Hybridization 1. Agarose:SeaPlaqueor NuSieveGTG (PMC BioProducts,Rockland,ME). 2. Electrophoresisunit for submarinegelsanda constantvoltagepowersupply (Holzel, Dorfen, Germany). 3. Gel trays (11 x 13 cm) and a 1Cteeth comb bridge (Holzel, Dorfen, Germany). 4. Nylon membrane(11 x 13cm) (BoehringerMannheimGmbH, Mannheim, Germany). 5. , Maidstone,England). 6. Glass tray and glass plates or alternatively a vacuum-blotting device (VakuGene,PharmaciaP-L Biochemicals, Milwaukee, WI).

05% xylene cyan01 FF. 7. DNA sequencing apparatus (including glass plates, spacers, and combs). 3. Methods 1. Digest the 5’ ends of the PCR product by mixing 2 pL of PCR product (from a 100 uL reaction), 2 pL of 10X Klenow buffer, 1 pL of Klenow fragment, and 15 PL of H20 in a microfuge tube. 2. Let stand for 10 min at room temperature. 3. 5 pL of [a-3*]dCTP and 1 pL of dNTP solution. 4. Let stand 10 min at room temperature (see Note 1). 5. Destroy the enzyme by incubating at 70°C for 5 min. 6.

And Collins, F. S. (1991) Construction of T-vectors, a rapid and generalsystemfor direct cloning of unmodified PCR products Nucleic Acids Res. 19, 1154. 15. Holton, T. A. and Graham, M. W. (1991) A simple and efficient method for direct cloning of PCR products using ddT-tailed vectors Nuclex Acrds Res. 19, 1156. CHAPTER 3 Direct Radioactive Labeling of Polymerase Chain Reaction Products Tim McDaniel and Stephen J. Meltzer 1. Introduction Radioactively labeled polymerase chain reaction (PCR) products are being used in an increasing number of molecular biology research techniques.

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Proceedings by ICM-1920, Strasbourg.

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