By Barbara A. Hamkalo and Sarah C.R. Elgin (Eds.)
A textual content meant for researchers and scholars in mobilephone biology, genetics and molecular biology, developmental biology, biochemistry and biophysics. The complicated challenge of the connection among nuclear constitution and serve as calls for a multidisciplinary, multifaceted method. This laboratory consultant is designed for researchers, from graduate scholars to professors, who want distinctive protocols and common discussions on a extensive variety of recommendations. the amount offers a variety of other methodological methods for the research of nuclear constitution and serve as - from cytological to molecular to genetic. those contain visualization of nucleic acid sequences utilizing hybridization probes, visualization of proteins utilizing immunological probes, isolation of chromatin fractions, mapping "in vitro" protein-DNA interactions, reconstitution of useful templates and nuclear substructures, and genetic techniques to settling on and characterizing chromosomal elements.
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Extra info for Functional Organization of the Nucleus: A Laboratory Guide
M. l% Tween 20. 5)]. Let stand for 10 minutes at room temperature. Fill the tube with IBT. Repeat steps c and d. n. Resuspend the pellet in IBT at lo* cells/ml. Count cells in hemacytometer after a 50-fold dilution of a sample of cells in IBT containing 2 pg/ml Hoechst 33258. The suspension should consist primarily of single, intact nuclei. 4. Denaturation and hybridization: a. Prepare the hybridization mixture by mixing 8 parts HB1 (see Section VI,A) with 1 part 10 mg/ml sonicated herring testes DNA.
Mix. Incubate at 37°C for 45 minutes. c. 05% Tween 20). Let stand with occasional mixing at room temperature for 10 minutes. d. Centrifuge as above, remove supernatant by aspiration, and flick pellet. e. Repeat step 6a. f. Add 20 pl of a 1 : 100 dilution of FITC conjugated goat anti-mouse IgG antibody (Sigma). Incubate at 37°C for 45 minutes. g. Repeat steps 6c and 6d. 7. Fluorescent labeling of biotin probes: a. 1% Triton X-100 and 5% BSA) to the pellet. Vortex gently to mix. Let stand for 10 minutes at room temperature.
Mix 100ml of complete medium and distribute 5 ml into each flask. The complete medium can be made up in advance and stored at -20°C. Growth medium contains RPMI 1640 medium + HEPES (Irvine Sci. cat. no. 9159) supplemented with 20% fetal bovine serum 1% penicillin/streptomycin (Irvine Sci. cat. no. 9366) 1% L-glutamine (Gibco, cat. no. 320-5030AG) 13 units/ml heparin (Sigma cat. no. H8514) 4% PHA (from a fresh hydrated stock made up by adding 10 ml of sterile distilled water to PHA from Gibco, cat.