By Curtis A. Machida
Oregon health and wellbeing Sciences Univ., Beaverton. offers a set of reproducible equipment 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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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.
The melting temperatures, Tms, for each primer should be within 1–2°C of each other (see Note 12). 5. The primer annealing temperature for PCR is approx 5°C lower than the Tm of the oligonucleotides (see Note 13). 6. The selection of primers from known sequences can be determined visually or with computer programs. 0 (National Biosciences, Plymouth, MN) or Prime from GCG (see Note 14). 7. Primers for amplifying DNA of more than 2 kb in length (long-distance PCR, LDPCR) are designed to have higher annealing temperatures to provide greater specificity.
Selection of Primers for PCR 1. Oligonucleotide synthesis facility. 2. Sequences of adrenergic receptor subtype genes. 3. 0 (National Biosciences, Plymouth, MN) or Prime (Genetics Computer Group [GCG], Madison, WI). 4. Computer programs for comparing DNA sequences: Pileup program (GCG). 5. PCR enzymes and reagents. 6. Thermal cycler. 4. Reverse Transcription and Selection of Primers 1. 2. 3. 4. 5. 6. 7. 8. Oligonucleotide synthesis facility. Sequences of adrenergic receptor gene subtypes. Computer programs for analyzing primers.