The SCE stain is done using the following protocol

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The SCE stain is done using the following protocol Powered By Docstoc
					The SCE stain is done using the following protocol SCE Stain Materials 1. Bromodeoxyuridine (BrdU) stock solution, 1 mg/ml in PBS, HBSS, or SF-DMEM. 2. Hoechst 33528 (bisbenzimide) stock solution, 10 mg/ml 3. McIlvaines buffer pH8 Sol.A-0.1 M Citric Acid 2.25 ml Sol.B-0.2 M Disodium phosphate 97.75 ml 4 6% Geimsa in Gurr's Buffer (Gibco/BRL) pH6.8 Procedure 1. Add BrdU to growing cultures to a final concentration of 10 ug/ml. Incubate for 2 cell cycles, 36 to 60 hours, depending on the cell line. 2. Harvest cells for metaphase chromosomes as usual. Do not dry the slides on a slide warmer or heat. 3. Stain slides in Hoechst 33528 (200 ug/ml) in water for 30 min. At room temp. Rinse well with water and air dry or blot dry, do not heat. 4. Place 100 ul of McIlvaines buffer on the slide and cover with a 22x60 mm coverslip. 5. Place slide on slide warmer at 55-60*C under long and short wave UV light for 20 min (we use a UV crosslinker). Rinse slide with water and blot or air dry . 6. Stain in Geimsa for about 10 min. From: Goto, K., S. Maeda, Y. Kano, T. Sugiyama. Factors involved in differential geimsa-staining of sister chromatids. Chromasoma. 66:351-359, 1978.


				
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