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Samford University -- Department of Biological and Environmental Sciences
Experimental Genetics   Biol 334
Karyotyping
Important Notes
1) Wear gloves. Each student will handle his/her own blood through the first addition of Carnoy's solution below. These steps are marked with a diamond ◊. This may mean some students must process multiple tubes up through that point.
2) If you spill Carnoy's solution on you, rinse it off immediately.
3) Mixing thoroughly with the pipette at various steps below is very important. Don't underdo it.
5) You should practice pipetting with a 10 mL pipette while waiting for the centrifuge.
6) Wipe down your work table with 70% alcohol before and after the lab exercise.

Culturing Cells
  • (Blood collected by venapuncture at Samford Student Health Services.)
  • Dispense 10 mL of PB-MAX Karyotyping Medium (from GIBCO) into a 25 mL vented culture flask.
  • Under sterile conditions, add 0.75 mL whole blood to the culture medium.
  • Incubate at 37C for about 68 - 72 hours. Use vented cap position of culture flasks.
  • About 1 hr. before harvesting cells, add 100 μL Colcemid (from GIBCO, Colcemid is the trademark name of a 10μg/mL solution of the spindle fiber inhibitor called colchicine).
  • Mix gently by inverting the flask several times.
Harvesting Cells
  1. ◊ After 68-72 hrs. of culture, close the lid tightly and mix the flask by gentle inversion until the cells are dispersed.
  2. ◊ Pour the contents of the flask into 2 15 mL conical centrifuge tubes (4 tubes if you are sharing).
  3. ◊ Centrifuge at 1300-2000 rpm for 5 min.
  4. ◊ Using a disposable pipette, remove the supernatant (upper liquid) down to the 0.5 mL mark, being careful not to disturb the rather fluffy pellet of cells at the bottom of the tube. Discard this supernatant in the Hazardous Waste container on your table. (You can reuse this pipette in subsequent steps.)
  5. ◊ By using the pipette bulb to suck and blow, mix (resuspend the cell) thoroughly. Do not blow bubbles.
  6. ◊ NOTE THE TIME. EVERYONE MUST BEGIN THIS STEP AT THE SAME TIME. Pour in about 10 mL 0.068M KCl and IMMEDIATELY mix THOROUGHLY by sucking and blowing with the pipette.
  7. ◊ Allow to sit at room temperature for 15 min. (During the wait steps, go to "Making Chromosome Spreads" step 2-4.)
  8. ◊ Pour in about 0.5-1.0 mL Carnoy's and mix THOROUGHLY with pipette.
  9. Add Carnoy's to the 10 mL mark and mix THOROUGHLY.
  10. Centrifuge for 5 min. at 1300-2000 rpm.
  11. Use the same pipette to remove supernatant (discard in Hazardous Waste beaker).
  12. Pour in about 10 mL Carnoy's and MIX THOROUGHLY with pipette.
  13. Let stand for 10 min.
  14. Centrifuge for 5 min. at 2000 rpm.
  15. Use the same pipette to remove supernatant (discard in Hazardous Waste beaker).
  16. Pour in about 5 mL Carnoy's and MIX THOROUGHLY with pipette.
  17. Fill to 10 mL and MIX THOROUGHLY with pipette.
  18. Centrifuge for 5 min. at 2000 rpm.
  19. Use the same pipette to remove supernatant (discard in Hazardous Waste beaker).
  20. Pour in about 2-5 mL Carnoy's (depending on the amount of cells you have) and MIX THOROUGHLY with pipette.
  21. Place on ice for 10 min.
Making Chromosome Spreads
  • Slides have been cleaned in 95% ethyl alcohol.
  • Remove 5-10 slides from the alcohol and clean them thoroughly with a KimWipe. Handle the slides by the edges only.
  • Handling the slides only by the edges place slides on a slide tray frosted side up and label the tray with your name. (
  • Place tray in the freezer for at least 30 min.
  • Take slides from freezer. If frost has formed on them, wait for it to thaw, then make spreads immediately while there is still fog on the slide. If there is no fog, breath on the slide to form fog and make spreads immediately.
  • Make spreads by dropping a drop onto the slide from  1 - 6 ft. above. Drop 2 to 3 drops on each slide, spacing the drops out horizontally along the slide.
  • Allow the spreads to dry thoroughly. (Blow on them, but do not spit. A hair dryer may be used, but you must be careful not to blow you slides away.)
Staining Slides
  • Quick Staining
    • Stain 4-5 min in Coplin Jar in 2.22% Giemsa (11.1 mL Giemsa + 500 mL Gurr pH6.8 buffer)
    • Holding the slides in place, pour the excess Giemsa in to the "Used Giemsa" container
    • In the sink, run water into the coplin jar, flushing out the stain, then continue rinsing under running water for about 3 min
    • Shake off excess water and place slides on slide tray to dry (hair dryer may be used)
  • Quick G-Banding
    • Place slides on 60-65C warming tray overnight.
    • Stain 4-6 min in Coplin Jar in 2.22% Giemsa (11.1 mL Giemsa + 500 mL Gurr pH6.8 buffer) + 110 μL 0.25% trypsin
    • Holding the slides in place, pour the excess Giemsa in to the "Used Giemsa" container
    • In the sink, run water into the coplin jar, flushing out the stain, then continue rinsing under running water for about 3 min
    • Shake off excess water and place slides on slide tray to dry. (hair dryer may be used)
Finding Chromosome Spreads
  • When slides are completely dry, observed them under a microscope, first under 100X, the 400X. When you find very good spreads, use 1000X and a drop of immersion oil. (No coverslip is necessary.)
  • Search for spreads that have 46, non-overlapping chromosomes.
  • I will photograph and give to each student a print of a good spread.
Making Karyotypes
  • Cut out individual chromosomes, leaving a small, white margin around each.
  • Don't throw away any scrap paper until your karyotype is complete.
  • Arrange chromosome in to groups A - G and sex chromosome, using your karyotype exercise guide.
  • Arrange chromosomes in each group from large to small.
  • Line up centromeres on the line.
  • Label each of the 7 groups and individual chromosomes.
  • Have your layout checked before gluing chromosomes down.