Institute for Hygiene and Microbiology, University of Wuerzburg
Manufacture protocol
oligonucleotides purchased from Operon Biotechnologies, Inc.
ARRAY PRINTING 1. Dissolve oligonucleotide probe or PCR product in the appropriate spotting solution to obtain the recommended final probe concentration: DNA Probes Final Spotting Concentration Oligonucleotides 10 - 20 micromolar PCR products 0.1 – 1 mg/ml 2. Transfer an appropriate volume of probes to a microtiter plate. 3. Set up the arrayer according to the manufacturer’s recommendations. If you were previously using slides that were thicker than 1.0 mm, for optimal spotting you may need to re-calibrate the distance between the slide surface and the spotting pins. 4. Print the substrates at 40-50% relative humidity at 20 to 25°C. DNA IMMOBILIZATION 1. Incubate printed microarray slides in humidity chamber at room temperature for 30 min (see appendix for details of how to prepare this chamber) to ensure quantitative immobilization. 2. Proceed to washing HANDLING OF PRINTED ARRAYS The volume of washing solution should be at least 250 ml per 5 slides. Make sure that slides do not dry out between washing steps, or between the washing and blocking step. WASHING 1. Wash slides to remove unbound probe molecules and buffer substances to avoid interference with subsequent hybridization experiments. a. Rinse 1 x 5 min in 0.1% Triton X-100 at room temperature. b. Rinse 2 x 2 min in 1 mM HCl solution at room temperature. c. Rinse 1 x 10 min in 100 mM KCl solution at room temperature. d. <optional> Denaturation step for arrays spotted with PCR-probes: 1 x 3 min in boiling diH2O. e. Rinse 1 x 1 min in diH2O at room temperature. 2. Proceed to Blocking immediately. BLOCKING 1. Block the slides with Nexterion® Blocking Solution or alternatively with 50 mM ethanolamine, 0.1% SDS (add freshly before use) in 0.1 M Tris, pH 9.0 as follows: a. Incubate slides 1 x 15 min in 1x Nexterion® Blocking Solution at 50°C. The volume of blocking solution should be at least 100 ml for 5 slides. b. Rinse 1 x 1 min in diH2O at room temperature. • Dry the Nexterion® Slide E in an oil-free air or nitrogen stream or by centrifugation (200 x g for 5 min) to avoid any water stains on the slide surface. • Proceed to hybridization. HYBRIDIZATION 1. Re-suspend the dried, labeled target to be applied to the array in Nexterion® Hyb. In case the target is already dissolved in a different buffer or in water, the sample can also be diluted in Nexterion Hyb to get at least 90% (v/v) buffer in the final hybridization solution. 2. Denature the suspended target by heating at 95°C for 3 min in a heat block, perform a quick spin in a microcentrifuge, then pipette the appropriate volume onto the array surface of a blocked slide under the coverslip or inside a hybridization station. POST-HYBRIDIZATION WASHING 1. Place the array into a slide rack and immerse in a dish containing 2x SSC and 0.2% SDS. Wash in the above solution 1 x 10 min at room temperature. 2. Wash 1 x 10 min in 2x SSC. 3. Wash 1 x 10 min in 0.2x SSC at room temperature. 4. Dry the array in an oil free air or nitrogen stream or by centrifugation (200 x g for 5 min) to avoid water stains on the slide surface. 5. Protect the array from light, dust and abrasion of the array surface, until ready for scanning. Ensure that the laser and filter set of the scanner is compatible with the fluorescent labeling of the probe molecules.
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