By Steven M. Powell
Instructs investigators in key genetic, mobile, and molecular organic equipment of studying colorectal tumors. suggestions and assays are defined intimately to permit researchers to scan on colon tumors. so much chapters are written through scientists who pioneered the equipment offered. insurance contains gross microdissection of colon tumors, institution of mobile traces and xenografted tumors, chromosomal analyses similar to FISH assays, mutational analyses of colon tumors and blood samples, gene expression analyses, and microsatellite instability trying out. contains a few colour pictures. For researchers at a number of degrees, from graduate scholars to pros. Powell is affiliated with the college of Virginia health and wellbeing procedure.
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Extra info for Colorectal Cancer: Methods and Protocols
3. Stop the reaction by heating the Eppendorf tube at 70°C for 10 min. 4. Add 400 ng (800 ng for paraffin-embedded tumors) of each of both labeled DNAs (tumor and reference of the same sex), 20 µg of unlabeled Cot-1 DNA, 10 µL of 3 M Na acetate and 700 µL absolute ethanol. Mix and keep for at least 2 h at –20°C to precipitate the DNA probe. 5. In a microcentrifuge at 4°C, spin the probe for 30 min at 10,000g. Discard supernatant and allow the DNA pellet to dry at 37°C for 20–30 min. 6. The DNA probe is dissolved in 10 µL of hybridization buffer for at least 2 h at 37°C.
Is the most wonderful feature offered by fluorescence in situ hybridization (FISH). DNA in situ hybridization is a technique that allows the visualization of defined sequences of nucleic acids within the individual cells. The method is based on the site specific annealing (hybridization) of single-stranded labeled DNA fragments (probes) to denatured, homologous sequences (targets) on cytological preparations, like metaphase chromosomes, interphase nuclei, or naked chromatin fibers. Visualization of hybridization sites becomes possible after detection steps by using a wide spectrum of the fluorescent dyes available.
2. Preparation of Metaphase Slides (see Note 2) 1. Adjust the humidity to 70–80% and the temperature to 25°C in a hood. 2. From a distance of 30 cm, drop one to two drops from your fixed cell suspension onto the slide. 3. Keep the slide 5 min inside the humid hood before taking it out. 4. Check your metaphases with a phase-contrast microscope and accept only slides with black spread metaphases. 5. Allow the slides to age at room temperature (25–30°C) for 1–2 wk. 6. Aged slides can be successfully used for another 2 wk.