Karyotyping 1st day short protocol وراثة عملي
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This document provides instructions for preparing metaphase chromosome spreads from cultured peripheral blood cells. It is a multi-step process that involves harvesting the cell culture, fixing the cells, making chromosome slides, and aging the slides. To make the slides, fixed cell suspension is placed onto a tilted slide and flooded with fixative to produce an even dispersal of cells across the slide surface. The slides are then examined under a microscope to check chromosome spreading and morphology.
Karyotyping 1st day short protocol وراثة عملي
- 1. AL Aqsa university Medical Technology department 1 Metaphase Chromosome Preparation from Cultured Peripheral Blood Cells Day 1 laboratory session: HARVEST CULTURE Required time 1. Add 50 μl of 10 μg/ml Colcemid to culture tube. Incubate 30 min in a humidified 37◦C, 5% CO2 incubator. 30 min 2. Centrifuge 7 min at 500×g, room temperature. Discard supernatant. 7 min 3. Add 5 ml of 75 mM KCl pre‐wormed at 37◦C and gently resuspend cells. Let stand 15 min at 37◦C. 15 min 4. Add 1ml of ice‐cold fixative drop by drop with a Pasteur pipet while mixing by vortex. Centrifuge as in step 5. 7 min 5. Remove all but 0.5 ml of the supernatant and resuspend pellet in remaining supernatant by drawing it gently up and down with a Pasteur pipet. Add 5 ml ice‐cold fixative drop by drop while mixing by vortex. Leave on ice for 20 min. Centrifuge as in step 5. 20 min 6. Aspirate supernatant, resuspend pellet and repeat step 8 (without incubation) until the pellet is clear. 7. Remove supernatant and resuspend pellet in a volume of fixative sufficient to produce a light milky suspension (about 0.5ml). Allow to stand 30 min at room temperature or store overnight at 4◦C. 30 min CHROMOSOME SLIDE PREPARATION 1. Remove slide from methanol and polish with lint‐free tissue. Dip slide once in methanol and then several times in deionized water until the methanol is gone and a thin, uniform film of water covers the slide. 15 min 2. Hold the slide as in the figure 3. Hold a Pasteur pipet in a horizontal position 1 to 2 inches above the slide. Place 3 drops of cell suspension, evenly spaced, onto the slide, as in the figure 4. Tilt the slide as in step 2 and flood with fresh 3:1 methanol/aceƟc acid fixative, dropwise, using a Pasteur pipet as in the figure. 5. Air‐dry the slide. 6. Examine slides for good chromosome spreading and morphology by phase‐contrast microscopy.
- 2. AL Aqsa university Medical Technology department 2 AGING SLIDES WITH HEAT Incubate air‐dried slide of metaphase chromosomes 2 days at 55°C or 20 min at 90° to 95°C (using dry oven or slide warmer). If incubaƟon Ɵme will be longer than 2 days (e.g., over a weekend), decrease temperature. Figure 1 Chromosome slide preparation: (A) After blotting the long edge of the slide to obtain a thin uniform layer of water, the slide is tilted to ∼30◦ and 3 separate drops of fixed cell suspension are applied starting away from and proceeding toward the frosted end. This sequence allows excess fixative and water to flood onto the frosted end without pooling on the slide. Application of the drops 1/3 of the distance from the top of the slide (indicated by Xs) counteracts the downhill dispersal tendency of cells on the slide and promotes even dispersal across the slide width. (B) After application of the cell suspension, the slide is flooded with fixative across the top edge, again proceeding toward the frosted end. This displaces a front of remaining water across the slide and onto the frosted end. It is important to avoid pooling of excess fluid on the surface of the slide, and to obtain a thin, even film of fixative to ensure uniform drying.