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.