Protocols in Human Molecular Genetics by Charles R. M. Bangham (auth.), Christopher G. Mathew (eds.)
By Charles R. M. Bangham (auth.), Christopher G. Mathew (eds.)
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Perform APCR for 35 cycles. Typically, denaturation is at 94°C for 40 s, followed by annealing at 55°C for 40 s. Primer extension by the TagDNA Wilson 34 polymerase then proceeds at 72°C for 90 s. These conditions are sufficient to amplify DNA inserts of up to 4000 bp in length. 5. 5mL microcentrifuge tube. 2, and 200 yL of isopropanol. Mix briefly and let stand for 30 min at room temperature. 6. Pellet the DNA by centrifugation at 13,OOOgfor 15 min at room temperature. tL of 70% ethanol, and dry briefly under vacuum.
Precipitate the heteroduplex once with ethanol, wash once with 70% ethanol, and dry. tL contains 1000 dpm. 4. Prepare a homoduplex labeled control plus unlabeled control identically for the control reaction. 2. DNA/RNA 1. 1-l pg) so that there is at least a 12x excess of RNA, precipitate the mixture, wash with 70% ethanol, and dry the pellet. 2. Add 40 l,tL of DNA/RNA annealing buffer. Incubate at 90°C for 5 min, then at 55’C for 2 h, and then ethanol precipitate. Wash the pellet with 70% ethanol and dry.
Boston, MA). Simple homemade systems for thermal cycling have also been described (8). The automated fluorescent DNA-sequencing system described here is model 373A, manufactured by Applied Biosystems Inc. The current configuration includes an Apple@ Macintosh@ IIcx computer with an 80-MB hard disk drive. Thermus aquaticus (Taq) DNA polymerase: This may be purchased from one of several enzyme suppliers; however, it is our experience that the enzyme supplied by Cetus gives the best results. For DNA-sequencing reactions, the enzyme of choice is the modified bacteriophage T7 DNA polymerase (US Biochemicals, Cleveland, OH) (9).