Hi,

Colchicine replaced by  Colcemide.

The final concentration of Colcemide should be 100 ng/ml. You can  Add 100 ul of 10 ug/ml colcemid to 10 ml of medium and incubate 1.5 hr more in 37oC.

To Trysinization, dissolve 0.12gr Trypsin   in 100 ml Versen buffer.  To do banding,  the slides must be aged and then  submerge  them in Trypsin solution for 30-45 Sec follow by staining. However, it must be  optimize based on  your own lab condition to get high quality banding. The longer colcemide treatment, the shorter Chromosomes.

Hi

For 3 ml of culture you can add 20 µl of colcimid at 69th hour and incubate it for 7 min then centrifuge the tube and add 4 ml of 0.56% KCl solution with simultaneous vortex and place it in water bath for 25min.

 KaryoMAX Colcemid Solution in PBS Catalog number: 15212-012

Concentration is 10µg/ml

In our lab we use both colchicine and colchicine for mitotic arrest of hyman blood lymphocyte cultures. Colcemid is a bit better, but colchicine also works OK, and we have been happy with it for 30 years of our practice. We use the work solution of 0,3 mg/ml colchicine in normal saline and put it as 20 (twenty) microliters into 5,5 ml of culture for the last 4 hours of culturing. Longer time of cell incubation in presence of colchicine leads to higher outcome of metaphases, but also to higher proportion of spreads with super-contracted chromosomes, which are unsuitable for G-banding. So, you should optimize the time and concentration by yourself. The work solution of colchicine, filtered through antibacterial syringe filter, can be kept in a fridge at +4 degrees Celsius for several months. The stock for work solution, prepared from the colchicine powder dissolved in a distilled water (usually 1000x concentration) is kept in a freezer for years. Good luck!

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Thank you all for the  answers...

Is there a difference in the protocol for leukemia samples? and what is it?