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以Zymolase處理yeasts, 抽提酵母菌DNA的方法?

XINXIFENLEI:SHENGMINGKEXUEZIXUN  RIQI: 2011-12-19 1:19:50

分割線

CHOUTIJIAOMUJUNDNA

YIXIAJIESHAOYIZymolaseCHULIyeasts, SHICHENGWEIspheroplasts, ZAIYISDSSHIPOLIE, ZAIYIpotassium acetateJIANGSDS, proteinsJIcellular debrisCHENDIAN, ERHESUANZEBAOLIUZAISHANGQINGYEZHONG, ZHEZHONGFANGFALAICHOUTIyeastDEDNA.

BG捕鱼CAI    LIAO:

YPD medium

Sorbitol solution

0.3mg/ml Zymolase (20,000U/g) in sorbitol solution

0.28M β-mercaptoethanol

Tris/EDTA solution

10﹪SDS

BG捕鱼5M Potassium acetate

BG捕鱼100﹪ethanol

TE buffer

1mg/ml RNase A

BG捕鱼100﹪isopropanol

BG捕鱼15ml LIXINGUAN(YONGGUOJIQISHI)

30℃JI37℃ZHENDANGPEIYANGXIANG

65℃SHUIRONGQI

FANG    FA:

1.     ZAI15mlWUJUNLIXINGUANZHONG, PEIYANG5mlDECHUNyeastJUNZHONG(YOUDANYIJUNQUNJIEZHONG),  30℃, ZHENDANGPEIYANG24hr, 350rpm, CISHIXIBAOSHUYUE1~2×108cells/ml.

2.     LIXIN3,000rpm, 5min, JIANGSHANGQINGYEXICHU.

BG捕鱼3.     JIARU0.5ml sobitol solution, CHONGFENHUNHE.

4.     JIARU5μlDE0.3mg/ml zymolase in sobitol solution, YIJI50μlDE0.28M β-mercaptoethanol, CHONGFENHUNHE(votex), ZHI37℃, 1hr, ZHENDANG200rpm.

5.     LIXIN3,000rpm, 5min, XICHUSHANGQING, JIARU0.5ml Tris/EDTAYE, JIANGYETIXISHANGXIXIAYICHONGFENHUNHE, ZAIJIANGQUANBUYETI(JIXIBAOFUXUANYE)ZHUANRU1.5mlDEWEILIANGLIXINGUANZHONG.

6.     ZAIJIARU50μlDE10﹪SDSYE, HUANHUANHUNHE, ZHI65℃, 20min.

7.     ZAIJIARU200μlDE5M potassion acetate, HUANHUANHUNHE, ZHIBINGSHANG30min.

8.     LIXIN10,000rpm, 3min.

9.     JIANGSHANGQINGYEZHUANRULINGYILIXINGUANZHONG, ZAIJIARU100﹪ethanolZHILIXINGUAN, HUANHUANHUNHE. (CISHIDNAHUICHUXIAN)

BG捕鱼10.LIXIN10,000rpm, 10sec, CHENDIANDNA, DAOCHUSHANGQING, JIANGDNARONGYU300μl TEZHONG.

11.JIARU50μlDE1mg/ml Rnase A, CHONGFENHUNHE, ZHI37℃, 1hr.

12.JIARU0.5mlDE100﹪isopropanol, HUANHUANHUNHE, ZHIDNACHENDIANCHULAI.

13.JIANGDNAYIWEILIANGXIGUANJIANTOUQUCHU, BINGZHUANRULINGYIXINLIXINGUANZHONG, JIARU125μlDETE buffer.

PEIFANG:

BG捕鱼Sorbitol solution:

0.9M sorbitol

BG捕鱼0.1M Tris,Cl, pH8.0

0.1M EDTA

Tris/EDTA solution:

50mM Tris,Cl, pH8.0

20mM EDTA

FUZHU:

ZAICHOUTIyeast DNASHI, plasmidsYEHUIYITONGXICHU, QILIANGDAYUESHIyeast DNALIANGDE1/1,000, YIJIZAI2mlDEyeast cellsKETICHUYUE4μgDEDNA, ERQIZHONGplasmidsYUEYOU4ng, KEZHIJIEYONGLAIZHUANZHI. (ZHIYAOYONG0.1μgDEDNAJIKE, QIZHONGYUEHAN0.1ngDEplasmeds)

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