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酵母菌基因转植的方法和步骤?

信息分类:生命科学资讯    作者:yiyi发布 

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MUQIANJIANGJIYINZHUANZHIRUyeastZHONG, ZHUYAOYOUDIANZIQIANG, DIANJI, lithium acetateJIspheroplastsDENGFA, CICHUJINJIESHAOlithium acetateFA. CIFASHIJIYUYANGLIZIKESHIyeastDEXIBAOMONENGSHIDNATOUGUO, ZAIJIARUDNAHOU, ZAIJIARUpolyethylene glycol(PEG), ZAITUZAIXUANZEXINGDEPEIYANGMINSHANG.

CAI    LIAO:

YPD medium

TE buffer

Lithium autate solution

5mg/ml carrier DNA (sheared E.Coli DNA)

PEG solution

HANXUANZEXINGHUAXUEPINDEPEIYANGMIN

FANG    FA:

1.     JIANGCHUNHUADEDANYIJUNQUNyeastJIARU2ml YPDYEZHONG, ZAI30℃CHENGZHANGYIWAN.

2.     JIANGXIBAOXISHIZHIOD600=0.2(YUE3×106cells/ml)QU50ml YPD(HANSHANGSHULIANGZHIyeast)YU250ZHUIXINGPINGZHONGSHIZHANG4hr, 30℃, SHIDAOD600=0.5~1.0(YUE1~2×107cells/ml), (SHIYONGSHIYUE10mlKEZUOYIGESHIYAN).

3.     JIANGXIBAOZHUANRUWUJUNLIXINGUAN, LIXIN2,500rpm, 10min, DAOQUSHANGQING, JIANGXIBAORONGZAI10mlDElithium acetateYEZHONG.

4.     ZAILIXIN2,500rpm, 10min, DAOQUSHANGQING, μlDElithium acetateYEZHONG.

5.     JIANGXIBAOFUXUANYEZHUANRUYIZHIXIAODUGUODE13×100mlSHIGUANZHI30℃, QINGQINGZHENDANG50rpm, 1hr.

6.     JIANG100μlDESHANGSHUXIBAOFUXUANYEJIARUWEILIANGLIXINGUANZHONG(YIGONGKEYOU5ZHI), CHUKONGZHIZUNEIZHIWAI, QIYUGEJIARU10μgDEDonor DNAJI20μgDEcarrier DNA(BAOZONGTIJIBUYAODUOYU15μl). DUIKONGZHIZUZEZHIJIARUcarrier DNA, JIANGYETIYIpipettorSHANGXIACHOUXIHUNHE. ZHI30℃, 30min.

7.     MEIZHISHIGUANJIARU0.7ml PEGYE, YISHOUZHIQINGDANSHIGUANHUNHEZHI, ZHI30℃, 45min, ZAIZHI42℃, 5min.

1.     JIANGMEISHIGUANNEIXICHU200μlTUXUANZEXINGPINGPAN, ZHI30℃, 2TIAN, KEKANJIEGUO.

2.     JIANGZAIXUANZEXINGPINGPANSHANGDEFENLIJUNZHONG, QUSHUGE, FENBIEPEIYANGYUPEIYANGYEZHONG, YIBIANBAOCUN.

PEIFANG:

lithium acetate solution:

0.1M lithium acetate

10mM Tris.Cl, pH8.0

1mM EDTA

YIGUOLVXIAODU

PEG solution:

40﹪(w/v) PEG3350

0.1M lithium acetate

10mM Tris Cl, pH8.0

1mM EDTA

GUOLVXIAODU

FUZHU:

CIZHONGFANGFAKEDEYUEMEIμg DNA, YOU1~10GEXIBAOKEZHUANZHICHENGGONG.

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