Southern Blotting

1. Depurination of DNA fragments: Wash gel in 0.25 M HCl 5' (small gels) 7' (large gels) 2. Denaturation: Wash gel in (0.5 M NaOH 1.5 M NaCl) for 20' (small) to 30' (large). 3. Neutralization: Wash in ...

Materials: Whatman 3 mm Blotting Paper nitrocellulose (Schleicher & Schuell Amersham) or nylon membrane filter (Amersham). Paper towels (preferably C-fold "cheap-o" variety) Pyrex or Tup ...

Day 1 1. Digest DNA for 6 hours (or overnight) BSA 10 mg/ml 0.5 22.65 μl of 10 μg Genomic DNA RnaseA 10mg/ml 0.1 in TE mixed to 7.35 μl cocktail Spermidine 100nM 0.75 per sample 10X enzyme b ...

Two protocols are given here. The first one is used for BAC library screening on filters although is also suitable for Transfer hybridizations. PREPARATION OF HYBRIDIZATION SOLUTION Prehybridizati ...

DNA是通过异羟基洋地黄毒苷（digoxigenin，Dig）配基标记的脱氧尿嘧啶核苷三磷酸（dUTP）随机插入结合而被标记。dUTP通过间臂连结类固醇半抗原异羟基洋地黄毒苷酸基，形成Dig-dUTP，杂交反应后，杂交的靶DNA通过酶联免疫法与一个抗体复合物结合，接着在5-溴-4氯-3-吲哚磷酸盐（X-磷酸盐）和硝基四氮唑蓝（NBT）存在下，由酶催化反应，在杂交部位形成蓝紫色带或颗粒 ...

Has anyone any experience of using a semi dry blotter for Southern blotting. At present I am tranfering overnight using a weight I think there is a blotter that is suitable for nucleic acids and can t ...

1) 取10μl待测DNA，于一定浓度的琼脂糖凝胶上进行电泳。(20mL，1.0%凝胶) 2) 凝胶用溴化乙锭染色，切掉凝胶四周多余部分，并在凝胶的一角作一记号， 拍照记录电泳结果(拍照时， 凝胶旁放一尺子)。 3) 杂交用胶的制备 (做以下步骤两组合一) A. 将凝胶浸没入30mL 0.25mol/L HCl溶液中15min，使DNA脱嘌呤。 B.用蒸馏水短暂洗凝胶2次。 C. ...

实验原理: Southern印迹是将DNA片断从电泳凝胶上直接转移至膜支持物（如硝酸纤维素膜、尼龙膜）上，使DNA片断固定的技术。先将DNA经限制性内切酶消化成一系列片段，进行琼脂糖凝胶电泳，各片段因分子量不同而彼此分开，然后经碱处理凝胶，使DNA的片段被变性、中和并通过毛细作用在高盐缓冲液中在原位将单链核酸转印到硝酸纤维膜上，烘干、固定。 试剂和器材 一、试剂 变性液：1.5mol/L NaCl ...

This kit contains materials for six groups to perform Southern transfer and hybridization analysis using the included lambda DNA samples and biotinylated probe. The intellectual objective of the exper ...

采用地高辛高效标记混合物(ROCHE) 标记DNA探针 将10 ng~3μg的DNA探针（基因组、质粒或基因片段）用蒸馏水稀释至16μl 煮沸10分钟使DNA变性，迅速放在冰上冷却，以防其重退火。 加入4μl地高辛高效标记混合物，振荡混匀 37℃下过夜培养 加入2μl 0.2M EDTA (pH 8) 使反应停止，并于65℃ 下10min加热降低其活性。 使用前煮沸探针10 ...

一 酶切和电泳 在200 μl 微量离心管中加入： 25 μl DNA样品(约10μg)， 3μl 限制性内切酶（MBI，10 U/ μl） 5 μl 相应的10×buffer， 补水到50μl。 然后加一滴矿物油覆盖 稍微离心后放于37℃水浴8-12小时。酶切完后，取5μl 酶切DNA样品于0.8%的琼脂糖凝胶上检测酶是否充分。如果酶切 ...

Hi can anyone answer this I am having problems with my southern hybridization. I am wondering if the DNA transfers to my membrane. Is it possible that the DNA can wash into the solutions when denaturi ...

1. Run gel ...

Southern Hybridization Protocols Two protocols are given here. The first one is used for BAC library screening on filters although is also suitable for Transfer hybridizations. PREPARATION OF HYBRID ...

Analysis of Genomic DNA by Southern Hybridization Select several independent BAC clones containing the same inserts that will be used as a probe. Confirm the BAC clone integrity using by comparing Hi ...

实验步骤： 1．取 10μl DNA 于 0.8% 凝胶检测； 2．将 DNA 调节浓度至 300-400 ng/μl； 3．仔细阅读将所用的任何一种酶产品说明书，熟悉反应条件及酶切的贮存浓度（ 10U-50U/μl ）厂家配套试剂； 4．计算据反应条件所需要的各种试剂准确用量：（ 0.5 ml tube 中） DNA(3-5μg)&nb ...

Dige ...

Analys of Genomic DNA by Southern Hybridization (Southern Blot) Outline: Localization of particular sequences within genomic DNA is usually accomplished by the transfer techniques described by South ...

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