作者compaq (不是你)
看板Biotech
标题[求救] Two-stage Cell transformation
时间Wed Mar 5 12:14:01 2008
想请问一下有人做过two stage cell transformation的实验吗
我是用MNNG以及TPA处理当作positive control
之後做soft agar assay看细胞有没有trasnformation
可是做出来都没有colony
不知道哪里出了问题
以下是我的protocol
1. Treat 2ug/ml of MNNG for 72hr
2. Remove MNNG containing media and culture cells for 3 days in normal
conditioning media without additives.(我是用serum free medium)
3. Culture cells for 2 more weeks in conditioning media containing
100ng/ml TPA.
4. Following TPA treatment, culture cells one more week in normal conditional
media, without assitives.
5. Continue with soft agar assay.
我的soft agar base layer是0.5%
top layer是0.33%
1000 cells/well(6 well plate)
cell line是mouse embryo liver cell
先谢谢大家的回答
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