DNA实验技术

Prepare: 200ml LB in a 1L flask 1L sterile ddH2O and place in cold room 4 50ml conicals chilled on ice 10% glycerol (cold) Chilled boxes of 100μl and 1ml pipet tips. Chilled 0.5ml tubes. General Th ...

1、 感受态细胞的概念 重组DNA分子体外构建完成后，必须导入特定的宿主（受体）细胞，使之无性繁殖并高效表达外源基因或直接改变其遗传性状，这个导入过程及操作统称为重组DNA分子的转化。 在原核生物中，转化是一个较普遍的现象，在细胞间转化是否发生，一方面取决于供体菌与受体菌两者在进化过程中的亲缘关系，另一方面还与受体菌是否处于一种感受状态有着很大的关系。 所谓的感受态，即指受体（或者 ...

DNA 分子是分子生物学研究的基本材料，依不同的实验目的可采取不同的抽提方法获取数量和质量不等的 DNA 。 实验目的：了解植物 DNA 抽提的主要方法，掌握 CTAB 法快速抽提水稻 DNA 。 实验材料及试剂： 水稻叶片，1.5 × CTAB ，氯仿 / 异戊醇 (24:1) ，95% 乙醇或无水乙醇等 实验原理：植物 DNA 的抽提常采用两种方法： （1）SDS 法： 离子去污剂 ...

一、目的 1.了解感受态细胞生理特性及制备条件，掌握大肠杆菌感受态细胞制备方法。 2.掌握质粒DNA 转化大肠杆菌的方法，了解转化的条件和利用半乳糖苷酶基因插入失活选择重组质粒DNA 的原理。 二、原理 （一）大肠杆菌感受态细胞制备的原理 所谓感受态，是指细菌生长过程中的某一阶段的培养物，只有某一生长阶段中的细菌才能作为转化的受体，能接受外源DNA而不将其降解的生理状态。感受态形成后，细胞生理状态 ...

Preparation of Silica 1. Suspend 5 g of silica (Sigma S-5631) in 50 ml of PBS. 2. Allow the silica to settle for 2h. 3. Discard the supernatant containing fine particulate matter. 4. Repeat steps 2 an ...

Sugden labMcArdle Laboratory for Cancer Research University of Wisconsin-Madison Medical School http://mcardle.oncology.wisc.edu/sugden/Protocols/html files/Filter Binding Assay.htm Last Modified 5.2. ...

DNA的克隆是指在体外将含有目的基因或其它有意义的DNA片段同能够自我复制的载体DNA连接，然后将其转入宿主细胞或受体生物进行表达或进一步研究的分子操作的过程，因此DNA克隆又称分子克隆，基因操作或重组DNA技术。DNA克隆涉及一系列的分子生物学技术，如目的DNA片段的获得、载体的选择、各种工具酶的选用、体外重组、导入宿主细胞技术和重组子筛选技术等等。 一 目的DNA片段的获得 DNA克隆的第一步 ...

本方法通过SDS裂解细胞，蛋白酶降解蛋白，CTAB去除多糖成分 一:仪器:同方法一 二:试剂: TE、TAE缓冲液;10%SDS;5MNaCL; 20mg/ml蛋白酶K;CTAB/NaCL溶液(10% CTAB，0.7M NaCL 4.1gNaCL 溶于80ml水中，缓慢加入10gCTAB，加热溶解);25/24/1，酚/氯仿/异戊醇; 24/1，氯仿/异戊醇;异丙醇;70%及100 ...

(adapted from Bruce A. Roe Department of Chemistry and Biochemistry The University of Oklahoma Norman Oklahoma 73019 broe@ou.edu) A. Sonication The generation of DNA fragments by sonication is perfor ...

Hi.- I have a problem with amplification of DNA by PCR. The extraction of DNA is direct from plant. i use PVP for keep out of DNA Polyphenols from the samples. The problem is: i can't view apmlificati ...

1.Wash the plates briefly using the 96-channel block washer. 2.Place the plates in a tub submerged in dd-water. 3.Put a flask on top of the plates to keep them submerged. 4.Place the tub with the s ...

The following protocol is based on our modifications of R. Kraft J. Tardiff K. S. Krauter and L. A. Leinwand. Biotechn . 6(6):544-545 1988. 1.Inoculate 2-5 ml of L broth containing the appropriate ant ...

Donis-Keller Lab Manual Department of Genetics in Washington University School of Medicine http://hg.wustl.edu/hdk_lab_manual/plasmid/plsmid13.html Purpose: To maintain lab stock of highly efficient ...

1.Remove gel slice contain DNA fragment and place in 10 volumes of: 300 mM NaOAcpH 7.0 300 ml 1 M NaOACpH 7.0 1 mM EDTA 2 ml 500 mM EDTApH 8.0 698 ml ddH20 2.Incubate at 22℃ for 30 min.Transfer gel sl ...

(adapted from Bruce A.RoeDepartment of Chemistry and BiochemistryThe University of OklahomaNormanOklahoma 73019 broe@ou.edu) A.Large scale double-stranded DNA isolation The method used for the isolati ...

This protocol was written by Jean-Pierre Issabased on Adams et al.* Kam-Wing Jair has made some useful shortcuts that work well if you are careful.Here is .This assay can be used to measure activity i ...

Does anybody know how to isolate single-stranded DNA from mammalian cells? Thanks for responding. Yin -- -------------------------------------------------------------------------------- Hello I don't ...

Recovery of DNA from LMP (Low Melting Point) Agarose Gel 1. Separate DNA fragments through an LMP agarose gel containing ethidium bromide (0.5 microgram/ml). 2. Detect DNA by irradiating the gel with ...

Alkaline lysis miniprep 1. Grow bacteria overnight in 37℃shaking incubator with lids very loose and taped on. I normally use 5 ml of liquid medium in a 50ml conical bottom tube. Be sure to include the ...

一、导论 质粒提取的原理:转自复旦大学一位老师的帖子后面是方法介绍 碱裂解法从大肠杆菌制备质粒是从事分子生物学研究的实验室每天都要用的常规技术。可是我收研究生十几年了几乎毫无例外的是我那些给人感觉什么都知道的优秀学生却对碱法质粒抽提的原理知之甚少。追其原因我想大概是因为《分子克隆》里面只讲实验操作步骤而没有对原理进行详细的论述。这是导致我的学生误入歧途的主要原因。后来我发现其实是整个 ...