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Fluorescein isothiocyanate-dextran solution

Chromosomal localization of the axl gene is done by fluorescence in situ hybridization (FISH). The biotin-labeled axl probe is from a cosmid vector containing a 41-kb genomic fragment. The slides with human metaphase cells for chromosomal DNA to be denatured are immersed into a solution of 70 % formamide-x4 in saline sodium citrate buffer (SSC) atpH 7.0 (0.15 M NaCl 0.015 M sodium citrate) at 70 °C for 2 min. For the slides, the hybridization mixture kept at 75 °C consists of 50 %formamide,xl SSC, 10 % dextran sulfate pH 7.0, and 150 mL unlabeled sonicated total human genomic DNA add 50-100 ng labeled probe. The mixture is incubated at 37 °C for 10 min. The slides are washed in 50 % formamide and SSC x3, 5 min each at 40 °C and in SSC x3 at 40 °C. The detection reagent consists of x4 SSC-0.1 % Triton, and 1 % bovine semm albumin for 3 washes 3 min each at 40 °C. The slides are incubated with fluorescein isothiocyanate-conjugated avidin at 37 °C for 30 min. Metaphase cells are counterstained with 4,6-diamidino-2-phenylindole dihydrochloride 200 ng/mL in 2x SSC for 5 min at... [Pg.182]


See other pages where Fluorescein isothiocyanate-dextran solution is mentioned: [Pg.105]    [Pg.181]    [Pg.323]    [Pg.350]    [Pg.193]    [Pg.186]    [Pg.3579]    [Pg.101]    [Pg.387]    [Pg.316]    [Pg.102]    [Pg.105]    [Pg.157]    [Pg.829]    [Pg.131]   
See also in sourсe #XX -- [ Pg.181 ]




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