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The mixture was then pipetted onto the microarray slide and covered.
Extension solutions were injected between the microarray slide and the cover slip and single base extension was allowed to proceed for ten minutes at 65°C.
Quantification of the eight sub-arrays of each microarray slide and 38 hybridized microarray slides resulted in approximately 910,000 data points.
The hybridization solution was injected between the microarray slide and the cover slip and hybridization reactions were incubated at 65°C for 10 minutes.
Labeled reference and test DNAs were combined in 45 µl Pronto! cDNA hybridization solution (Corning, Corning, NY) and heated to 95°C for 5 min. Then, 15 µl of the hybridization mixture was put onto a microarray slide and sealed with a cover slip.
Labelled cDNAs were simultaneously hybridized to a microarray slide and detected with Cy5 (biotin) and Cy3 (fluorescein), respectively.
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We thank Bjørn E. Kristiansen, the Norwegian Microarray Consortium, Oslo, for printing the microarray slides and Linda H. Godager for performing the real-time quantitative RT-PCR.
Hybridization to microarray slides and washing steps were performed as described by Wang et. al., 2005, except that hybridization was conducted at 55°C.
After labeling and fragmentation, RNA was added to hybridization buffer (100 µl total volume, 6X SSPE, 0.05% Tween-20 [Sigma], 20 mM EDTA [Ambion], 25% formamide [Ambion], 100 ng/µl salmon sperm DNA [Sigma], 0.05% SDS [Ambion]) heated at 95°C for 3 minutes, chilled briefly on ice, added to microarray slides, and incubated at 45°C overnight.
RNA samples were randomly allocated to microarray slides and sectors.
XK and JM provided the prostate tissue microarray slides and evaluated the stained slides.
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