DNA实验技术

RNA labeling (Amersham Pharmacia)For the generation of radiolabelled single stranded RNA for use as hybridization probes 32P-pCp 3' End-labeling RNA (Jim Brown Lab)This procedure includes two metho ...

・ RNA Gel (Crawford Lab)・ Gel Electrophoresis of RNA (Beverly Faulkner-Jones)great tips on RNA gel electrophoresis.・ Northern Gel and Transfer (gopher://iubio.bio.indiana.edu ...

・ cDNA Synthesis (Crawford Lab)mRNA can be converted into DNA (copy DNA cDNA) by annealing oligo-dT to the 3' poly-A tail that occurs on all eukaryotic mRNA. After the dTs bind to the As we wi ...

Primer extension analysis is used to determine the location and quantitate the amount of the 5´-end of specific RNAs. An end-labeled oligonucleotide is hybridized to RNA and is utilized as a primer b ...

mRNA末端快速扩增（RACE）Rapid Amplification of mRNA Ends by PCR (RACE) (PMCI Research) ...

Quantitative Determination of Peptides by Sulfhydryl (-SH) Groups New (Contributed by David Van Horn Dept. of Chemistry UC Berkeley Greg Bulaj Dept. of Biology University of Utah)This is the standard ...

蛋白质电泳（主要内容如下）One-Dimensional SDS-PAGE Two-Demensional SDS-PAGE Protein Electrophoresis in Agarose Gel Gel Staining Recipes One-Dimensional SDS-PAGE ・ Protein Gel and Staining (Gottschling Lab ...

Gene Mapping (Amersham)DNA extraction PCR amplification and gel resolution. ...

A Simplified System for Rapid Generation of Recombinant AdenovirusesGive detailed guide to the construction of recombinant adenoviruses. ...

High Resolution Genetic Footprinting (Stanford)Genetic footprinting is a technique for high-resolution mapping of the functional organization of a cloned gene. An in vitro transposition reaction with ...

・ Yeast Transformation (Gietz Lab)LiAc/SS-DNA/PEG Transformation・ Yeast Transformation (Breeden Lab)LiAc method・ Large-Scale Yeast Transformation (Yale Yeast Analysis Center)De ...

Yeast Hartwell's Complete (HC) Media (Gottschling Lab) Yeast Complete Media Yeast Media (PMCI) Yeast Media Solutions and Stocks (Donis Keller Lab) HC Dropout Plate Supplementation Table (Gott ...

Yeast DNA Preparation Yeast Genomic Preparation (Gottschling Lab)Rapid method for yeast genomic DNA isolation Yeast DNA Preparation (rapid glass bead prep) (Hahn Lab) Yeast Genomic DNA Preparatio ...

利用聚合酶链反应制备放射性标记的DNA探针1. 在一个0.5 ml的薄壁微量离心管中设置下列扩增/放射性标记反应体系：10×扩增缓冲液 5.0μl10 mmol/L dNTP溶液 1.0μl0.1 mmol/L dCTP 1.0μl20μmol/L 正向寡核苷酸引物 2.5μl20μM反向寡核苷酸引物 ...

用T4标记的5’末端1．无菌的1.5ml为了离心管置冰浴上顺序混合下列组分：50 pmol合成的寡核苷酸2.5μl 10×反应缓冲液10μl γ-PATP（33pmol）1.5μl T4多核苷酸激酶（15U）灭菌双蒸水加至25μl2．37℃温育30min。3．当反应物正温育时，以2000×g离心2min制备Sephadex G-25自旋柱。4．加入2μl 0.5mol/L EDTA或加热至68℃ ...

应用激酶交换反应的末端标记1．在置冰浴中的1.5ml微量离心管内依次加入：50 pmol合成寡核苷酸2.5μl 10×反应缓冲液1.5μl ADP（300μmol/L终浓度）10μl γ－PATP（33pmol）1.5μl T4多核苷酸激酶（15U）蒸馏水加至25μl2．37℃水浴温育30分钟。3．加入2μl 0.5mol/L EDTA或加热至68℃ 10min终止反应。混合液经Sephadex ...

Northern杂交1．在10~20 ml 预杂交液中68℃温育膜2h。2．若使用双链探针，在100℃下加热32P标记的双链DNA 5 min，使之变性，然后迅速移至冰水中冷却。3．把变性的或单链放射性标记的探针直接加到预杂交液中，在合适的温度下继续温育12~16h。4．杂交后，将膜从塑料袋中取出，在室温下迅速转移到含有100~200ml 1×SSC，0.1%SDS的塑料盒内，将盒盖好，置于水平振 ...

组织印迹的Northern杂交1）硝酸纤维素膜或尼龙膜上的组织印迹1．在塑料板上放置两层Whatman 1号滤纸，在滤纸上方放上一张普通纸，然后铺上一层尼龙膜。2．用双面刀片从植物组织如大豆的茎上切取一块切片（厚度约为1mm）。如果组织表面是湿的，则在Kimwipes纸上轻轻吸干或先用蒸馏水洗涤几秒钟然后再用Kimwipes纸吸干。用镊子将组织切片转移到硝酸纤维素膜上，注意一旦将切片放置在膜上就不 ...

DNA点杂交l DNA斑点印迹的制备预备1．冰浴中以70W左右的超声波处理基因组DNA 1min，用琼脂糖凝胶电泳检测片段大小，这些片段大小均应为7～10kb。2．用TE缓冲液将DNA稀释至0.5μg/μl。斑点印迹的制备1．加热5μg DNA（在10μl TE缓冲液中）至95℃ 10min，冰浴5min。用微量离心管短暂离心收集液滴，置冰浴。2．裁一张大小合适的滤膜，用铅笔在滤膜 ...

RNA印迹杂交1） Northern印迹的制备预备：1．按下述步骤，每泳道加10～20μg总RNA或0.5～1μg poly(A)+RNA进行甲醛/琼脂糖凝胶电泳，将凝胶放在紫外线透照仪上，旁边置一标尺进行拍照。a. 总RNA（10～20μg）用下列溶液在65℃温育5min：总RNA（10～20μg）6.0μl甲酰胺12.5μl10×MOPS缓冲液2.5μl甲醛溶液（37％）4.0μl ...