Colorectal Cancer: Methods and Protocols (Methods in by Steven M. Powell

By Steven M. Powell

The contents of Colorectal melanoma: equipment and Protocols goal to show investigators in the entire key genetic, mobile, and molecular organic tools of reading colorectal tumors. The concentrated strategies and assays are defined in adequate element to permit researchers to begin an test on colon tumors and continue from commencing to finish as though the specialist within the box who has played those reviews have been guiding them on the bench. Of observe, many of the chapters during this quantity are written by way of these scientists who p- neered those tools and assays of their respective fields. The chapters in Colorectal melanoma: tools and Protocols describe “state of the artwork” how to research colorectal tumors, starting from gross mic- dissection of specimens to express molecular analyses. integrated are coverages of mutational assays, instability trying out, immunohistochemical assays, chro- somal reports, and gene expression analyses. The target of our quantity is to facilitate the functionality of colorectal tumor organic experiments by way of investigators at a number of degrees of training―from graduate scholars and postdoctoral fellows to central investigators who wish to boost our figuring out of colon melanoma improvement.

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Example text

B-Cells Culture 1. Grow cells in 10 mL RPMI-1640, with penicillin/streptomycin and L-glutamin and 15% FBS. 2. Split the culture 2 d before harvesting. 3. Change the media again next day, leave overnight (see Note 3). 3. Harvesting 1. Add ethidium bromide (8 µg/mL) for one hour. 8 mg/mL) per 10 mL media. 2. After 1 h add colcemid (10 µg/mL) 100 µL to 10 mL flask and mix well by shaking or pipeting. 3. Transfer to 15 mL tube immediately. 4. Incubate at 37°C for 15 min. 5. Centrifuge for 5min at 1000 rpm.

Fetal bovine serum (Gibco-BRL). Phytohemagglutinin (PHA) (Murex Biotech Ltd , Dartford, England, cat. no. HA15). Ethidium bromide, 10 mg/mL, (Gibco-BRL, cat. no. 5585UA). Colcemid, 10 mg/mL (Boehringer Mannheim, cat. no. 295892). Methanol (JT Baker, cat. no. 9093-03). Glacial acetic acid (Malinckrodt, cat. no. UN2789). 2. Reagents for Slide Processing 1. 1% Tween-20), store at room temperature up to 1 mo. 2. 70%, 85%, 100% Ethanol (–20°C) (VWR, cat. no. MK70194). 3. Deionized sterile water. 4. Pepsin, 10% stock solution (Sigma, cat.

Hybridization Incubate overnight at 37°C in a moist chamber. For CGH continue hybridization for 3 nights. 5. Washing 1. Remove rubber cement with a forceps. Soak slides in 2X SSC solution at RT. Quickly remove coverslips. Do not dry the slides. All washes are done at 45°C preferably in a shaking water bath. 46 2. 3. 4. 5. Pack and Zhuang Wash in 50% formamide/2X SSC, twice for 7 min. 1X SSC twice for 5 min. 1X SSC twice for 5 min (see Note 7). 1% Tween-20, 5 min at RT. During the following steps slides should be kept wet and protected from light!

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