I have performed several alterations in the G banding protocol for human bone marrow samples. However, I still can not see a stable banding pattern on chromosomes. Bands are somewhat dispersed along the chromosomes. I need a good protocol that works stable.
Thanks to anyone who may provide a protocol for the g banding of human bone marrow.
Well . I am using 13mg of Trypsin in 100ml PBS which gives me a proper band.I got it from Vellore manual.Just try it out.
We usually use 2-5mg of trypsin in 0.9% NaCl for the bone marrow sample.Give it a try!
In clinical diagnosis, we usually use 0.25mg of trypsin (Gibcol) in 100ml of EDTA, pH6.5- 7.0. But you still need to try the best digestion time for the chromosomes. Temperature and humidity will influence the results
Dear Kuei-Fang Lee
Many thanks for your response. We use 0.2 g trypsin with 80 mL PBS. However, I think that the main problem is with the aging of slides. Could you please tell me about how many hours you incubate your slides in the heat chamber? Or do you perform another way of slide aging? Because I believe that aging is a very critical step in G-banding and is associated with good bands along the chromosomes. We incubate the slides at 60 C overnight and than stain with giemsa. But this protocol is not so fruitful most of the times.
Thank you
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