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 advanced challenge of the connection among nuclear constitution and serve as calls for a multidisciplinary, multifaceted procedure. This laboratory advisor is designed for researchers, from graduate scholars to professors, who desire distinctive protocols and common discussions on a vast diversity of recommendations. the amount offers a variety of other methodological techniques 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 ways to determining and characterizing chromosomal elements.
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Extra resources for Functional Organization of the Nucleus: A Laboratory Guide
Exp. Cell Res. 153, 61-72. Landegent, J. , Dirks, R. , and van der Ploeg, M. Use of whole cosmid cloned genomic sequences for chromosomal localization of by non-radioactive in situ hybridization. Hum. Genet. 77,366-370. Langer-Safer, P. , and Ward, D. C. (1982). Immunological method for mapping genes on Drosophila polytene chromosomes. Proc. Natl. Acad. Sci. A. 79,4281-4385. Lawrence,J. , and Singer, R. H. Quantitative analysis of in situ hybridization methods for the detection of actin gene expression.
For photography, add an equal volume of anti-fade solution (see Section VI,D) to the nuclei suspension before covering with a coverslip. c. See Section VI,E for excitation and emission filter information. d. Photograph as described for slide hybridization (Section V1,E). VIII. Conclusions The procedures for DNA sequence localization in interphase and metaphase cells described in this chapter have a number of research applications. Several of these applications are illustrated in Color Plate 1. , 1989).
Use a Speedvac centrifuge to remove remaining ethanol from pellet. 15. Resuspend pellet in TE to achieve a DNA concentration of 100ng/pl. 16. Measure optical density (OD) of DNA at 260 nm. 0 at 260 nm. Adjust concentration as necessary by dilution or ethanol precipitation (step 14). 17. Check DNA size by agarose gel electrophoresis to determine if sonicated fragments are 200-400 bp. , 1989). If fragments are still large, repeat steps 11-16. - IV. Blocking DNA Preparation Overview. An excess of unlabeled DNA is added to the hybridization reaction for two purposes: (1) to reduce nonspecific binding of labeled probe DNA to chromatin, cell residue, or glass surfaces.