Journal: Journal of Biomolecular Techniques : JBT
Article Title: A Rapid Method for Manual or Automated Purification of Fluorescently Labeled Nucleic Acids for Sequencing, Genotyping, and Microarrays
Figure Lengend Snippet: Effects of unincorporated dye precursors on fluorescent nucleic acid analyses. A, B: Comparison of DNA sequencing analyses performed without (A) or with (B) purification of unincorporated dye terminators. Sequencing reactions were prepared from 250 ng pGEMZf(+) plasmid and 4 pmol M13 −47 primer, using BigDye version 3.0 at a 2:10 (μL BigDye:μL total volume) reaction mixture with Better Buffer diluent. Purification B was performed with the RapXtract II Kit. DNA sequences were analyzed on an ABI PRISM 3100 Genetic Analyzer using POP6 polymer and injection parameters recommended for RapXtract Kits (2 kV, 45 s). C, D: Comparison of cDNA-microarray hybridization analyses performed without (C) or with (D) purification of unincorporated dye-labeled nucleotides from the labeling reaction. Labeled cDNA was prepared from 1 μg Saccharomyces cerevisiae mRNA using the CyScribe First Strand Labeling Kit and Cy3-labeled dUTP. Purification D was performed with the RapXtract Fluorescent Nucleic Acid Purification Kit. The cDNA was then hybridized to a yeast collage array at 67°C for 12 h. The microarray was visualized on a ScanArray Lite scanner at a laser output of 80%, PMT gain of 80%, and spatial resolution of 50 μm.
Article Snippet: Reactions were prepared using a CyScribe First Strand Labeling Kit (Amersham Biosciences, Piscataway, NJ) and 1 μg Saccharomyces cerevisiae messenger RNA (mRNA) (BD Biosciences Clontech, Palo Alto, CA) or 2 μg mouse brain RNA (Ambion, Austin, TX).
Techniques: DNA Sequencing, Purification, Sequencing, Plasmid Preparation, Injection, Microarray, Hybridization, Labeling, Nucleic Acid Purification