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用于修饰DNA的酶与使用方法

信息分类:生命科学资讯    作者:yiyi发布 

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YONGYUXIUSHIDNADEMEIYUSHIYONGFANGFA

MEIYINGBAOCUNZAI-20℃LENGJUZHONG, SHIYONGSHIQUCHUFANGZAI0℃BINGSHANG, MEIDEJIAGEANGGUI, YIZHENXISHIYONG.

GEZHONGMEIJUNYOUQITEBIEDEHUANCHONGYE. TONGCHANGJUNKEZAICAIGOUSHIXIANGCHANGSHANGSUOQU.

The Klenow fragment:SHIE.Coli DNA PolymeraseIKAOCDUANDEBUFEN,WEISHANQUYOUNH2DUANDE323GEANJISUANERCHENG,JUYOUDNA PolymeraseYU3'→5'exonucleaseDEGONGNENG. YIKlenow fragmentLAIJIANGHANYINGGUANGBIAODINGDEd NTPHUOFANGSHEXINGBIAODINGDEd NTPJIAYUDNAPIANDUANWEIDUAN, KEJIANGYUE0.1~4μgYOUDIANYONGZHONGTICHUNDEDNA, JIASHANGYINGGUANGBIAODINGDEd NTP, ZAIJIASHANGWEIBIAODINGDE3 d NTPs, ZAIJIASHANG1UDEKlenow fragment, ZAIJIARUSHILIANGbuffer, ZHI30℃, 20minJIKE(QIZHONGd NTPDENONGDUZAI0.5mM). CIYUANLISHILIYONGXIANZHIMEIQIEGEHOUZAIJINGDIANYONGTICHUNDEDNA, ZAIERDUANJUNYOU ----____DANGUBUFEN, Klenow fragmentHUIJIANGd NTPJIAYUDANGUCHU, CISHIXUZHUYIDESHISUOQUEDEDANGURUOZHENGHAOWUKEPEIBIAODINGd NTPDEHEGANSUANZEWUFABIAODING, GUYINGZHUYI. RUYIBan HIQIEGEDEHUIYOUGATCPAIXU, RUOYIEco RIQIEGEDEZEHUIYOUAATTPAIXU, CISHIRUOYIYOUBIAODINGDEGTPHUOCTPLAIBIAODINGZEWUXIAO.

YIKECAIYONGRandom Oligonucleotide-Primed synthesisFANGFALAIBIAODINGDNA, QIYUANLIWEI:JIANGDNAYIMOUYIXIANZHIMEIQIEGE, ZAIDIANYONG, QUCHUSUOXUDEDNAPIANDUAN, JIARESHICISHUANGGUDNAFENWEIDANGU, ZAIJIANGRandom Oligonucleotide(TONGCHANGWEILIUGEnucleotides)JIESHANGQU, ZAIFANGRUd NTPsJIKlenow frament, RUCI, ZEDNAPIANDUANJIUHUIBEIBIAODINGLE.

FANGFA:

1. ZAIYIGEXIAOXINGLIXINGUANZHONGJIARU2.5μlZHI0.5mM 3dCTPs.

2. ZAIJIARU10XZHIKlenow fragment buffer, 2.5μl.

3. JIARUYUBIAODINGZHIYOUBIAODINGdNTP, 1μl.

4. ZAIJIARU1μlZHIKlenow fragment.

5. ZAIYILINGYIZHILIXINGUAN, JIARUYUE0.03~0.1μgDEDNA(XIANGBEIBIAODINGDEDNA), JIRandom hexanucleotide(1μg), ZAIJIARUT.E. bufferSHIZONGTIJIWEI18μl, JIANGCILIXINGUANZHIFEISHUIZHONG3min, ZAIQUCHUFANGBINGSHANG.

6. JIANGERZHILIXINGUANWUHUNHE, ZHISHIWEN, 3hr, FANYINGJIWANCHENG.

Taq DNA PolymeraseZHISHIYONG:

CIMEIXIYOUThermus aquaticusJUNSUOFENLICHULAI, TADEZUIYOUXIAOWENDUSHIZAI75~80℃, TADUOBEIYONGYUPolymerase Chain Reaction(PCR). QISHIYONGFANGFAZAIJIESHAOPCRSHIZAIXINGJIESHAO.

RNaseYIYOUYOUBUTONGLAIYUANFENLICHULAIZHE, SUOQIEGERNADEWEIZHIYIBUTONG, DANYIBANRNaseJUNNENGZAIDUOZHONGFANYINGQINGKUANGXIACHANSHENGZUOYONG, QIESHIYONGHOURUOYAOCHUQUTA, TONGCHANGYAOJIARUProteinase K, ZAIYIPhenol extractionsJIethanol precipitationCHULI.

DNA ligases:

YONGYIJIEHEDANGUYOUQUEKOUDEDNAHUOSHUANGGUKEJIAOCHAPEIHEDEDNA(JIYIXIANZHIMEIQIEGESUOXINGCHENGDE ----____ JIAOCHAPIANDUAN. SHIXIASHIYONGDEligase, YAOATP, E.Coli ligaseYAONADWEINENGYUAN.

SHIYONGFANGFA:

1. ZAIWEILIANGLIXINGUANZHONGJIARUligaseHUANCHONGYE,ZAIJIARU0.5mM ATP, 1μg DNA(JISUOXUJIEHEDEDNA), ZAIJIARU1μDET4 ligase, ZHI15℃, 2hr, KECHENG.

2. RUOXIERDUANDUNDUANDNAJIEHE(JIWUJIAOCHAPIANDUANZHIDNA), ZEXU10BEIYISHANGDEMEIFANGNENGDACHENG. YOUYANJIUXIANSHIJIARUJIWEILIANGDEPEG8000KEZHUligaseDEGONGNENG.

YUNYONGligaseGOUJIANJIAJIEDNAFENZI:

1. ZAIJIANGXIANGGOUJIANDEDNAPIANDUANJINGDIANYONGYOUTICHUNZHIHOU, YIXIAFAJINXING:

2. QU0.1ZHI5μgXIANGJIEHEGOUJIANDEDNAFANGRUWEILIANGLIXINGUANZHONG(TIJIYI9μlWEIZHUN), ZAIJIARU10μlZHI2X ligase buffer, ZAIJIARU1μlZHI10mM ATP, ZAIJIARU20ZHI500μDET4 DNA ligase(YIYOUJIAOCHAPEIDUIDEDNAWEIZHUN).

3. ZHI15℃, 3hr, HUOGEYE, KECHENG.

4. JIANGCIJIEHEZHIJIAJIEMEIJIE, YIJIYINZHUANZHIFANGSHI, ZHIRUE.ColiSUZHU, BINGJIANCHAJIEGUO, YImarkerBIAOXIANQINGXINGKEZHIYOUWUZHENGQUEDEJIEGUO, RUOYOU, ZEJIANGHANCIZHENGQUEJIAJIEMEIJIEDEE.ColiJIAYIPEIYANG, ZAIYITICHUNZHITIFANGSHIHUOQUDALIANGSUOYAODEJIAJIEMEIJIE.

2X T4 DNA ligase bufferZHIPEIFANGWEI:

100mM Tris.Cl, pH7.5

 20Mm MgCl2

 20Mm DTT(dithiothreitolCIWUBIXULENGDONGBAOCUN)

ZAIGOUJIANJIAJIEMEIJIESHI, BIXUKAOLVDAOTAYAONENGZAISUZHUZHONGNENGZIXINGFUZHI, QIENENGJIANGSUOYAODEJIYINBIAOXIANCHULAI, ERQIEHAINENGJIASHANGmarker, YIBIANYOUmarkerZHIYOUWULAIJIANCEGOUJIANZHICHENGGONGYUFOU. ZAISHIYONGligaseSHI, CHANGHUIYOUGEZHONGJILVDEJIEHE, ERCHANSHENGFEISUOYAODEJIEGUO,YIYOUDUODUANJIEHEZHE, DANYINJIEYUHUORANLVYOUGUAN, LILUNSHANGRENGHUICHUXIANRUOGANSUOYAODEJIEGUO.

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