Lab Report Genetic Experiment 1&2
Name: Momoe Nasuno
Student ID: 10301016066
Major: M.B.B.S.
Group: 4
Date: 2011/11/24
Objective:
The objective of these two experiments is to first prepare metaphase chromosomes from culturing cells
and then stain the metaphase chromosomes with Giemsa to elicit a banding pattern throughout the
chromosome arms, designated G-Bands.
Procedure:
For experiment 1:
1. Culture cells for about 2-3 days with medium.
2. Add Colcemid (final concentration 0.07 ug/ml) and mix well. Incubate for 4 hour at 37°C before
collecting.
3. Collect the cells to a centrifuge tube and centrifuge at 1500 rpm for 8 min. Remove medium
completely except for about 0.5 ml of supernatant remaining above the cell pellet.
4. Resuspend the cells in the remaining medium and carefully add approximately 2 ml of prewarmed
(37°C) 0.075 M KCl, drop-by-drop, while agitating gently. Add an additional 6 ml of KCl, mix well.
5. Incubate for 20 min at 37°C in the waterbath.
6. Add 1ml freshly prepared fixative (Methyl alcohol/glacial acetic acid, 3:1), mix well.
7. Centrifuge the cells (1500 rpm for 8 min), remove the supernatant.
8. Resuspend the cells and fix the cells by adding 8ml of fixative; the first 2 ml should be added dropwise
while agitating gently.
9. Leave it for 10-15 min at room temperature, centrifuge the cells and remove the supernatant.
10. Centrifuge the cells and resuspend the cells in a small amount of fixative (4-5 drops) and drop the
suspension (2-3 drops) onto a cleaned cold microscope slide. Dry over alcohol burner. Mark the side of
the slide which the cells have been dropped.
11. Dry for 3-7 days at 37°C.
For experiment 2:
1. Dissolve 25mg trypsin in 50 ml 0.85% sodium chloride, warm the solution in waterbath at the
temperature of 37°C, add 2 drops of 0.4% Phenol-sulfonphthalein, and adjust pH to 6.4-6.6 with 5%
sodium bicarbonate.
2. Dip oven-dried slides from last experiment in the trypsin solution for 3-7 seconds.
3. Rinse slides in water.
4. Use a graduated tube to mix 0.5 ml Giemsa and 5 ml PBS buffer just prior to staining. Stain the slides
for 8-10 minutes in a coplin staining jar.
5. Rinse slides in water and air dry.
6. Observe the slides using a Microscope: first use low magnification lens, then switch to high
magnification lens; when using 100X magnification lens, dip cedar wood oil on the slides to help for
observation. (Clean with xylene after use)
Result:
Discussion:
In this experiment, we observed metaphase chromosomes under microscope using G-banding technique.
G-banding technique is used in cytogenetics to produce a visible karyotype through the staining of
condensed chromosomes. The chromosomes must be treated with trypsin before they are Giemsa
stained. The treatment of trypsin must be just right; it cannot be over trypsinized or under trypsinized.
Over-trypsinized chromosomes will be fuzzy and under-trypsinized chromosomes will produce bands
that are hard to distinct. Under the 100X magnification lens, with the help of cedar wood oil, I can
clearly see the chromosomes. But the bands cannot be seen clearly, it does not show the contrast bands
on the chromosome arm. This is probably because of the chromosomes are over trypsinized. (Or simply
because the picture is not clear enough to show)