By Guy B. Faguet
VA clinical middle and the scientific collage of Georgia, Augusta. stories tools most beneficial for the analysis and administration of hematologic malignancies. equipment are offered intimately for the professional researcher and the fewer skilled. DNLM: Lymphoma.
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Extra info for Hematologic Malignancies: Methods & Techniques
9. Microtome (when processing paraffin-embedded tissue). Xylol (100%). Methanol (100%). Sodiumisothiocyanate (NaSCN, 1 M). 5. Proteinase K stock solution (20 mg/mL). 5). CIA (24 parts chloroformϺ1 part isoamylalcohol). Chloroform (100%). 48 10. 11. 12. 13. 14. 15. Baudis and Bentz Isopropanol (100%). 2. Ethanol (biograde, 70%). 0. Sephadex G50 columns. Photometer for measurement of DNA concentration. 3. Nick Translation 1. 5 mg/mL bovine serum albumin (BSA). 2. 1 M β-Mercaptoethanol. 3. 375 mM dTTP).
The supernatant is then removed and again ice-cold fixative is added equally slowly as in step 8. This step is repeated at least once. 10. The tube is incubated on ice for 30–60 min. Afterward, it is spun again in the cooled centrifuge and the pellet is resuspended. The cycle of very slowly adding fixative-spinning-resuspending is repeated at least five times. 11. After the last cycle, the pellet is resuspended in about 1–2 mL fixative solution. 12. , above a heated water bath (95–100°C). One drop will provide a sufficient number of metaphase spreads for a CGH experiment.
Subsequently, the tubes should be cooled for some minutes on ice, before preannealing of the probes is performed for about 20 min at 37°C. At this point, the tube can be kept on ice. While performing the steps described above, prepare the slides containing the metaphase spreads in parallel. When using frozen slides, it is important to thaw them slowly, thereby avoiding water condensation. Slides stored at –80°C are first transferred to –20°C for at least 30 min. Then they are kept for at least another 30 min at +4°C, before unpacking them and keeping them at room temperature.