(2) After 4 to 5 days later when cells are in the log phase of growth,
add colcemid (0.02ug/mL) to the medium already in the flask
(3) After 6 hr, remove medium gently, add 5mL 0.25% trypsin and
incubate 10 min.
(4) Centrifuge in trypsin and discard supernatant trypsin.
(5) Resuspend the cells in 5mL of hypotonic solution (0.075M kcl) and
leave for 20min at 36.5C (for human cells use 37C)
(6)Add equal vol of freshly prepared ice-cold acetic methanol, mixing
constantly, then centrifuge at 100g for 5 min.
(7) Discard the supernatant mixture , mix the cell pellet with a
vortex mixer, and slowly add fresh acetic methanol with constant
(8)Leave 10 min on ice
(9) Centrifuge for 5min at 100g
(10) Discard supernatant acetic methanol and resuspend cell pellet
with vortex in 0.2mL acetic methanol, to give a finely dispersed cell
suspension with no visible clumps.
(11) Draw one drop into the tip of a glass pipette and drop on a cold
slide. Let the drop run down the slide as it spreads.
(12) Dry off rapidly using a hotplate at at 40C
(13)Examine cells and make more slides