By Curtis A. Machida
Oregon overall healthiness Sciences Univ., Beaverton. offers a set of reproducible tools for the examine of regulators of CNS-mediated habit and neural functionality. For neurobiologists, neurochemists, neurologists, and pharmacologists. DNLM: Receptors, Adrenergic - research.
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40 Scofield, Deupree, and Bylund 3. 1. Cosmid Genomic Library Screening 1. Titer the cosmid library by making the appropriate serial dilutions in cold LB/ kanamycin (25 µg/mL) media and plating the dilutions on LB agar/kanamycin (25 µg/mL) plates. The plates are incubated overnight at 37°C and the colonyforming units/mL (CFU/mL) are determined (see Note 1). 2. Mix the cosmid library, and remove 16 aliquots such that each aliquot contains 1 × 105 colonies/1 mL. Place the aliquots in separate microcentrifuge tubes.
Blue/white screening can be used to identify the recombinant plasmids. The colonies can also be picked with a toothpick, and a PCR reaction directly performed on the minute amount of bacteria using primers that flank the insert or gene-specific primers. The sizes of the products are then identified by gel electrophoreses (15). DeFrancesco (16) gives a complete discussion of the PCR-based cloning kits that are available for cloning these products. The investigator should keep in mind that PCR products generated by polymerases with 3′ to 5′ exonuclease activity, such as Pfu, will automatically create blunt ends that must be blunt-end-cloned or must be changed to single nucleotide overhangs in an extension reaction with Taq polymerase for TA cloning.
Perez, D. , Piascik, M. , and Graham, R. M. (1991) Solution-phase library screening for the identification of rare clones: isolation of an α1D-adrenergic receptor cDNA. Mol. Pharmacol. 40, 876–883. 14. , and Oshita, M. (1995) Functional subclassification of vascular α1-adrenoceptors. Pharmacol. Commun. 6, 23–28. 15. , and Muramatsu, I. (1991) Three distinct binding sites for [3H]-prazosin in the rat cerebral cortex. Br. J. Pharmacol. 104, 961–965. 16. Ford, A. P. D. , Daniels, D. , Chang, D. , Gever, J.