DNA制备

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 ...

RNA点杂交1） 纯化的RNA的点杂交和狭线杂交安装印迹装置1．切一张合适大小的带正电荷尼龙膜。用铅笔标上表示方向的记号。用水把膜简单弄湿，在20×SSC中室温泡1 h。2．在膜浸泡期间，先用0.1 mol/L NaOH小心清洗印迹装置，再用无菌水洗干净。3．把两片厚滤纸用20×SSC浸湿，再放到真空器顶部。4．把样品槽插入装置的上部，把湿尼龙膜放在样品槽加样孔的底部，用吸管在膜的表面滚动以去除 ...

Western杂交l 组织印迹的Western杂交在硝酸纤维素膜上制备组织印迹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，然后铺上一层硝酸纤维素膜。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 ...

　　　常规杂交反应由于受到探针解链温度、溶液中靶序列的初始浓度及探针长度的影响。样品中不同探针所对应的靶序列的拷贝数不尽相同，探针的解链温度也难以保持一致，这样不同位点的杂交速度并不完全与各自靶序列的拷贝数成正比，检测结果也就不具有良好的平行性。所以必需选择最理想的条件以尽可能使正确配对的序列不被遗漏，并使杂交错配降到最低。DNA双链进行杂交配对时，A:T间形成2个氢键而G:C间形成3个氢键，所以 ...

Purification of plasmid DNA (miniprep) with high yields using diatomaceous earthKyung-Soo Kim and Charles K. PallaghySchool of Botany La Trobe University Bundoora Vic 3083 AustraliaCorrespondence to C ...

Solublization of RNA in Formamidecontributed by James McCaughern-Carucci Yale University Resuspending RNA in Formamide (as reported by Chomczynski et al. Nucleic Acids Research) has several benefits o ...

Serum Protein Electrophoresis Tricine/Polyacrylamide Gel ElectrophoresisUsed for pilin processing analysis but generally useful for resolution of small (15-35 Kdal) proteins of similar size. SeeStrom ...

・ Easy YAC Preparation Method (Andrew DaviesShaw lab)・ Screening YAC libraries (Donis Keller Lab)This is a method for screening YAC libraries for specific DNA sequences. Transformants ...

CONTENTTransformation-Competent E. coli preparation Rubidium chloride method Inoue "ultra-competent" method Cosmid packaging protocol DNA Ligation and Transformation Protocols Rubidium Chloride m ...

Genome-wide Gene Expression Analysis (Richard Young Research GroupWhitehead Institute for Biomedical Research)A genoe-wide gene expression analysis using high-density oligonucleotide arrays Restri ...

・ Yeast Two-Hybrid System (Finley Lab)This is one of the most comprehensive and detailed guide to yeast two-hybrid system technique with introduction to its background. The following procedure ...

Carbohydrate Assay (Hancock Laboratory) (Accessible only by IE)This protocol is used to determine the relative amounts of LPS CHO present in a given strain. The assay can be done on one set of sample ...

Preparing Overnight Bacteria Culture (LaboratoryExperiments.com)This is a basic procedure for high school students and useful for those who are new to molecular biology. Bacteria Growth and Culture ...

Antibiotic Concentration in Media (Lazo Lab) ...

Gram Staining (+\-) (William H. Heidcamp) Gram-Staining Procedure (MEDIC U of Texas)Very nice and detailed method description for Gram staining Acid-Fast Stain (Putt's Method)To detect the presenc ...

Preparing Lawn Cells for M13 Cloning (Life Technologies)Lawn cells require the F' episome for M13 infection and may be prepared Streaking Lambda Phages (Donis Keller Lab)Phage are streaked onto a me ...

・ Lambda DNA Preparation (Stanford DNA Sequence & Technology Center)Detailed protocol for lambda DNA preparation with recipes・ Isolation of Lambda DNA: Quick Method (Molecular Genetics ...

Or gel mobility shift assay gel shift assay gel retardation electrophoretic mobility shift assay (EMSA) EMSA Using Oligos (Mike A. Dyer)Anneal two complementary oligos to make ds oligos as the probe ...