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PCR是应用一种由高温生存细菌的DNA合成酶

信息分类:生命科学资讯    作者:yiyi发布 

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DNALIANSUOHECHENGFANYING

PCRSHIYINGYONGYIZHONGYOUGAOWENSHENGCUNXIJUNDEDNAHECHENGMEI, Taq DNA Polymerase, JIASHANGPrimersJIdNTPs, ZAINENGKONGZHIWENDUXUNHUANDEHECHENGYIZHONG, XIANJIARESHIYUANBENZHIDNAYOUSHUANGGUFENCHENGDANGU, ZAIJIANGWENZHIPrimersKEYUDANGUDNAJIEHE, ZAISHENGWENZHIDNAHECHENGMEINENGCHONGFENYUNZUO, YIDUANSHIJIANHOU, YOUSHENGWENSHIDNAFENWEIDANGU, ZAIJIANGWENSHIPrimersJIEHE,ZAISHENGYIDIANWENSHIDNAHECHENGMEINENGCHONGFENYUNZUO, RUCIBUDUANXUNHUAN, YUE20~30CIZHIHOU, DNAJIUKEJIANGTEDINGPIANDUANBEIFUZHIDASHUBAIWANCI, RUCIYONGYIZHENCEWEILIANGDNAHUOCEDNAZHONGYOUWUTESHUPAIXUDENG, YINGYONGSHIFENGUANGFAN.

CAILIAO:

10XJI1X PCR reaction buffer

8mM dNTP mix

Oligonucleotide PCR Primers (SHANGXINGYUXIAXINGERGU)

DNAMOZI

5μ/μl Termus aquaticus DNA Polymerase (Taq Polymerase)

Mineral oil

a thermal cycler

FANGFA: 

1. ZAIWEILIANGLIXINGUANZHONGJIARU:

10μl 10X PCR reaction buffer

10μl 8mM dNTP mix solution

10μl 10μM SHANGXING(Upstream) PCR Primer

10μl 10μM XIAXING(Downstream) PCR Primer

59μL template DNA(1μg in nuclease free dH2O)

1μl Taq Polymerase(2.5μ/μl)

RUOTIJIYINGEXIANGCHENGFENDENONGDUZHIGUWEIDA100μl KEYI1X PCR bufferDIAOZHENG.

2. JIARU100μl mineral oil(FANGZHIZHENGFA)

3. SHEDINGthermal cycler, SHIXIANJIAREZHI94℃, SHIDNAFENCHENGDANGU, 2min.

4. ZAISHEDINGDNAFUHEWENDU, CIWENDUSUIPrimerZHIZUCHENGERBUTONG. DAZHIKEYIXIALIEJINGYANGONGSHITUIGU:

 Tm=81.5+16.6(logM)+0.41(GC﹪)-(500/n)

QIZHONGn=PrimerDEZHANGDU

MSHIHUANCHONGYEZHONGDEYANFENNONGDUMOERSHU(BUJIMg++DENONGDU)

LIRU, NaClNONGDUWEI0.04, TrisNONGDUWEI0.67, ZEWEI:

   0.04(NaCl)+0.67×0.01(Tris)=0.047M salt

RUOYI25GEbpDEPrimerERYAN, QITmWEI

   Tm=81.5+16.6(log0.047)+0.41(60)-(500/25)=64

ERTONGCHANGFUHEWENDUWEITm+22DU=66DU. DANRUOYISHIWEIBIANGUSUAN, ZEYI55℃JINXING, YIBANYEKECHENGGONG.

JINXINGYIFENZHONG.

5. ZAISHEDINGDNAHECHENGWENDU75℃, 2min. ZAI2FENZHONGSHIJIAN, DNAZHISHAOKEHECHENG1,000bp.

6. RUCISHEDINGthermal cyclerXUNHUAN3~5GUOCHENG3CI.

7. QUCHU10μl(QUZAImineral oilXIACENGYETIBUFEN), YIDIANYONGJIANSHIJIEGUO.

YIXIAJIANGGEYOUGUANCAILIAOCANKAOZILIAOLIECHUGONGBUSHIZHIXU(YIBANJUNXIXIANGCHANGSHANGZHIJIECAIGOUSHISUOQUHUODIAOHAOZHE, CICHUWEIRUOXUZIXINGDIAOPEISHICANKAOYONG)

*8mM dNTP:JIANG2mMZHIMEIZHONG(GONG4ZHONG)dNTPHUNRU, BAOCUNZAI-20℃.

*Oligonucleotide PCR Primers:YIdH2ODIAOCHENG10Um, BAOCUNZAI-20℃.

*10X PCR buffer:50mM KCl  100mM Tris.Cl, pH8.4

  15mM MgCl2200μg/ml gelatin

*Taq DNA polymerase:TONGCHANGYI5μ/μlNONGDUCHUSHOU, ERSHIYONGSHIMEI100μl, ZHIYAO1.5~2.5μJIZU, GUZAISHIYONGQIANKEYIPCR bufferDIAOZHENGNONGDU. CIMEIWUproof readingGONGNENG, YIWEIMINGXIANFAXIANYOUexonucleaseDEGONGNENG. ZUISHIYIDEFANYINGWENDUWEI75~80℃.

*ZAIPCRFANYINGZHONG, XIALIETIAOJIANBIXUZHUYI:

1. ZHISHAOYAOYOU2μTaq polymerase/100μl reaction

2. 1.5mMZHIMg+2/0.8mM dNTP mix

3. 100 Pmol each Oligonucleotide PCR Primers

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