Methods for analysis of nucleic acid methylation status and methods for fragmentation, labeling and immobilization of nucleic acids
a nucleic acid and methylation status technology, applied in the field of methods for analysis of nucleic acid methylation status, and methods for fragmentation, labeling and/or immobilization of nucleic acids, can solve the problems of increased cancer risk, increased methylation of dna, and associated risks, and achieve the effect of reducing the number of incubations
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Acid-Catalyzed Fragmentation and Labeling of cDNA
[0274] Single stranded cDNA was prepared from universal human total RNA (Stratagene, La Jolla Calif.). Pooled purified cDNA product at a concentration of 145 μg / mL in water was aliquoted into each of five 200 μL PCR tubes, dispensing 13 μL or 1.89 μg into each tube. 12 μL water was then added to each tube, followed by 2.5 μL of 0.5 M glycolic acid buffer (prepared by dissolving glycolic acid in water at a concentration of 1.0 M, adjusting pH with 1 M NaOH, then diluting to 0.5 M with water). Three tubes received buffer at a pH of 3.0, and two received buffer of pH 3.5. The pH 3 tubes were heated at 95° C. for 5 minutes and 65° for 5 minutes or 30 minutes. The pH 3.5 tubes were heated at 65° C. for 5 minutes or 30 minutes, all in a MJ Research Peltier Effect thermal cycler. Tubes were then held briefly on ice for the next step.
[0275] To each tube was added 5 μL of 0.5 M acetate buffer pH 4.33 (prepared by pH adjustment of acetic acid...
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