By Curtis A. Machida
Oregon future health Sciences Univ., Beaverton. presents a set of reproducible tools for the research of regulators of CNS-mediated habit and neural functionality. For neurobiologists, neurochemists, neurologists, and pharmacologists. DNLM: Receptors, Adrenergic - research.
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2. In other procedures, the products are blunt-ended, and cloned by filling the recessed ends with Klenow polymerase or by removing the extended bases with Pyrococcus furiosus (Pfu) DNA polymerase. 3. Finally, vectors with T overhangs can provide a sticky end for the A overhang in the PCR product in a procedure called TA cloning. This is one of the more popular methods for cloning PCR products and is described by Trower (9) (see Notes 25 and 26). 4. The identity of the PCR products can be confirmed by sequencing, Southern hybridization with known probes, or restriction endonuclease analysis (1).
23. 24. 25. 26. 5 µM oligo dT primers) and ~1 µg total RNA. After incubating at 42°C for 50 min and then at 95°C for 5 min to denature the enzyme, the entire mixture is amplified by PCR in a 50-µL reaction with the appropriate sense or upstream primer and PCR reagents. In comparison to random primers, oligo dT will tend to copy a greater proportion of the 3′ end of the mRNA, and random primers will transcribe more efficiently at the 5′ region of the mRNA. The success of this approach is dependent on how specific the antisense primers are at the lower-temperature conditions of 42°C for cDNA synthesis by reverse transcriptase.
A. (1994) Use of agarose block DNA to make cosmid libraries. Biotechniques 17, 278,279. 50. , Kingston, R. , Moore, D. , Seidman, J. , Smith, J. , et al. (1988) Plating lambda phage to generate plaques, in Current Protocols in Molecular Biology, vol. 1, Wiley, Interscience, New York, pp. 4. Receptor Gene Isolation 37 2 Isolation of Adrenergic Receptor Genes Margaret A. Scofield, Jean D. Deupree, and David B. Bylund 1. Introduction In order to isolate a single gene, phage or cosmid libraries can be screened by the conventional technique of hybridization as described by Sambrook et al.