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Boekel Scientific rapidFISH Hybridization Oven Used to Study the Effects of Humidity on a DNA Labeling System
Date:9/28/2018

Purification
The fluorescent-dye labeled bacmid DNA must be purified prior to hybridization. The DNA probe was purified using the QIAquick® PCR Purification Kit. Samples were gently mixed with 275 µL of provided PB buffer (Qiagen), applied to in the column and centrifuged at 16,100 x g for 1 minute at room temperature. Flow-through was discarded and 750 µL of provided PE buffer (Qiagen) was added for second centrifugation. DNA was eluted by using 10 µL of EB buffer (Qiagen), allowing the mixture to stand for one minute, followed by a centrifugation for one minute at 16,100 x g.

Determining DNA Fluorescent Dye Incorporation
The Thermo Fisher Nanodrop® ND-1000 UV-VIS Spectrometer was used in the Microarray Measurement Mode to determine the incorporation of Green 496 dUTP in each prepared batch.

Hybridization & Scanning
Human male metaphase slides from Abbott (Prod. No. 06J96-001) were warmed to room temperature and prepared for hybridization (2 µL from Batch 1 or 0.5 µL from Batch 2). Purified Green 496 probe and 3 µg (1 µg/µL) Cot-1 DNA were added to each sample. Varying amounts of nuclease-free water were added to the oven tray for each hybridization. All samples were immediately placed into the Boekel RapidFISH Slide Hybridization Oven for overnight incubation at 37 °C.

Hybridization quality was tested at 0 mL, 5 mL, 10 mL, 15 mL, 20 mL, 25 mL, and 30 mL. Each volume was tested in duplicate. A temperature and humidity data logger (Madgetech) was used to measure operating temperature and relative humidity at a sampling rate of 30 s. After incubation, each sample was washed and carefully stained using 8 µL of VECTASHIELD® H-1200 mounting medium containing DAPI (1.5 µg/mL) with antifade.
The hybridized samples were scanned immediately after hybridization and washing using an Olympus BX51 Fluorescence

Microscope with a 60X objective lens. All images were collected using 1 s exposure with both DAPI and FITC fluorochrome filters to best assess the effects of humidity on hybridization for FISH while minimizing photobleaching.

Results
Green 496 fluorescent dUTP was sufficiently incorporated into the bacmid DNA using the Nick Translation DNA Labeling System 2.0. Hybridization for FISH using the Boekel Scientific RapidFISH Slide Hybridization Oven yielded bright results with as little as 5 mL added moisture. Results were similar for moisture levels greater than 20 mL.

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