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将真核细胞的细胞核与细胞其他部分分开

信息分类:生命科学资讯    作者:yiyi发布 

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FENLIXIBAOHEJISHU

JIANGZHENHEXIBAODEXIBAOHEYUXIBAOQITABUFENFENKAI, YILIQITASHIYANZHIYONG.

CAILIAO:

Lysis buffer

[PEIFANG] 10mM Tris.Cl, pH7.43mM CaCl2

2mM MgCl2

Nonidet P-40(NP-40) lysis buffer B

[PEIFANG] 10mM Tris.Cl, pH7.43mM CaCl2

2mM MgCl2  1﹪NP-40

   (QIANSANXIANGPEIHEXIANGAOWENGAOYAXIAODUHOU, DAILENGQUEZAIJIARUNP-40)

JUNZHIQI

FANGFA:

1. DUIDANCENGZUZHIPEIYANGXIBAO, KEXIANDAOQUPEIYANGYE, YI5ml, 4℃ PBSQINGXIXIBAOERCI, ZAIYIGAOWENGAOYAXIAODUGUOZHIRUANJIAOGUADAOJIANGXIBAOGUAXIA, BINGLIXIN1,500rpm, 5min, 4℃; RUOWEIFUXUANXIBAO, ZEZHIJIELIXIN1,500rpm, 5min, 4℃, BINGYI4℃ PBSXIXIBAOERCI(LIXIN, DAOQUSHANGQING), BENSHIYANJUNYI5×107XIBAOLIANGSHEJI.

2. JIANGXIBAORONGYU15μl, 4℃, lysis bufferZHONG, HUANHUANHUNHE, 10min.

3. LIXIN4℃, 1,500rpm, 5min, DAOQUSHANGQING, ZAIJIANGXIBAORONGYU1ml lysis bufferZHONG.

4. ZAIJIARU1ml NP-40 lysis buffer B, HUANHUANCHONGFENHUNHE.

5. 将细胞吸入均质器, 搅动10次, 取少数液置血液计数器, 在显微镜下检视细胞是否只剩细胞核.

6. JIANGZHISHENGXIBAOHEDEXIBAORONGZHUANRUWUJUNLIXINGUANZHONG, LIXIN1,500rpm, 4℃, 5min.

7. ZIXIXICHUSHANGQING, JIANGCHENDIANZHIXIBAOHEZHIBINGSHANG, ZAIJIARU200μlZHIGANYOUBAOCUNYE(PEIFANG:50mM Tris.Cl, pH8.3

40﹪ glycerol

5mM MgCl2

0.1mM EDTA),

SHIXIBAOHERONGYUYEZHONG, ZAIJIANGZHIBAOCUNZAI-70℃ZHONG.

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