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Yeast的嫁接媒介的方法和步骤

信息分类:生命科学资讯    作者:yiyi发布 

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YeastDEJIAJIEMEIJIE

YOUXIEyeastDEJIAJIEMEIJIE(cloning vector)WEIplasmids, CHENGWEIYRP(yeast replicating plasmids), QIZHONGHANYOUyeast DNAFUZHIQISHIDIANDEPIANDUANJIYIN, CHENGWEIARS(autonomous replication sequences), DANZHEIZHONGplasmidsZAIFANZHIGUOCHENGZHONGCHANGBUNENGBAOCUNZAIXINDEXIBAOZHONG, RUOZAICIplasmidsZHONGJIARUCentromeres(CEN elements), ERXINGCHENGYCP(yeast centromeric plasmids), JIUKESHIQIWENDINGXINGZENGJIA.

LINGYOUYEP(yeast episomal plasmids), SHIYOUyeastDEplasmidSUOGOUCHENG. HAIYOUYLP(yeast linear plasmids)SHIZHIXIANXINGDE, YINHANYOUtelomeres, HAIYOUyeast artificial chromosome(p YAC), KEGONGJIAJIEYUE400KbDEDNA. CIplasemidBINGKEZHANGZAIE.ColiZHONG.

ZAIZHEIXIEJIAJIEMEITIZHONG, WEIBIANYUJIANCETEDINGJIYINDEGONGNENG, CHANGZAICITEDINGJIYINJIASHANGLac 2 gene, CIJIYINXIYINE.ColiZHONGDELAI, HUICHANSHENGβ-galactosidase, DANGCI  YUDAOYIXIELEISIβ-galactosidesWUSHI, HUIFENJIEZHIERCHANSHENGYOUYANSEDEFENJIEWU, HENRONGYIZHENCE. WEISHICIJIYINNENGZAIyeastZHONGYUNZUO, ZAICIJIYINQIANXUXIANJIASHANGyeastDEpromoter region, YIXIAJIESHAOYIZHONGCEβ-galactosidaseLIANGDEFANGFA.

CIFASHIJIYUHANCIJIYINDEyeast, ZHANGYUPEIYANGYEZHONG, JIARUONPG(O-nitrophenyl-β-D-galactoside), SHIZAI30℃ZUOYONG, ZAIYIDIDINGSHIpHDA11ZHONGZHIFANYING, ZAIYIGUANGPUYICESEDUBIANHUAERJISUANβ-galactosidaseDELIANG.

CAI    LIAO:

YPDPEIYANGYE

Z buffer

0.1﹪sodium dodecyl sulfate (SDS)

chloroform

4mg/ml ONPG in 0.1M KPO4, pH7.0 (GUOLVXIAODU, BAOCUN-20℃)

1M NaCO3

30℃ Water bath

FANG    FA:

1.     YIYIGEJUNQUNDECHUNyeastJIEZHONGYU5mlDEYPSYEZHONG, GUOYE(KEZUOER, SANGEJUNQUNYIBIANBIJIAO).

2.     YIGUOYEPEIYANGDEyeastYE50μlJIEZHONGWUJUNDEYPDYE5ml, SHIZHANGZHIOD=2.0(DAYUE0.5~1×108cells/ml).

3.     LIXIN2,500rpm, 5min, (ZHUOSHANGXINGLIXINJI), JIANGCHENDIANWURONGYUDENGLIANGZ bufferZHONG. (RUGUOβ-galactosidaseLIANGBUDUO, KEYIRONGYUJIAOSHAOLIANGDEZ bufferZHONG).

4.     YOUSHANGSHUBUZHOUZHONGZHIyeast, QUCHUERGEYANGBEN, YIWEI100μlXIBAOJIA900μl Z buffer, LINGYIWEI50μlXIBAOJIA950μl Z buffer.

5.     ZAIMEIYIYANGBENZHONGGEJIARU1DIChloroformJI2DI0.1﹪SDSYE, vortex 10sec, ZAIFANGRU30℃SHUIYUZHONG15min.

6.     ZAIMEIYIYANGBENZHONGJIARU0.2mlDE4mg/ml ONPG, vortex 5sec, BINGYU30℃SHUIYUZHONG, BINGKAISHIJISHI.

7.     FAXIANYOUHUANGSECHUXIANSHI, JIARU0.5mlDE1M Na2CO3BINGJIXIASHIJIAN.

8.     LIXIN5min, 2,500rpm, ZAICESHANGQINGYEDEOD420YUOD550(RUOSHANGQINGYEZHONGBUHANXIBAOPOLIEWU, ZEOD550TONGCHANGWEI0, KEYIBUJISUAN).

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