遗传学实验技术

Attractene Transfection Reagent 高效转染各种贴壁真核细胞 ♦ 高效DNA转染，极低细胞毒性 ♦ 快速、简便的操作流程 ♦ 所有贴壁细胞 （包括难转细胞）的最佳选择 ♦ 非常适用于瞬时转染，稳定转染，共转染和 shRNA载体转染 ♦ 不含来自动物的组分 更高效率转染质粒DNA Attractene 试剂采用非 ...

This protocol is based upon protocols from Mark Biggin Dave Allis and Richard Treisman plus a fair amount of trial and error. We have successfully used this protocol with livers spleens colons and who ...

Chromatin IP Method Day 1 1. Start 5 ml o/n cultures of strains to be tested Day 2 1. Inoculate 50 ml of the desired media with a volume of a saturated o/n culture and grow o/n with shaking at 200 ...

Many methods have been developed to identify genomic targets of DNA-binding factors. Here we outline and discuss our adaptation of the chromatin immunoprecipitation (ChIP) assay to a high-throughput m ...

染色质免疫沉淀后，多种PCR方法可分析共沉淀下来的DNA。定时定量PCR、二重PCR、hot-stop和SSCP是4种有效方法。

染色质免疫沉淀法（Chromatin immunoprecitation，ChIP）是研究体内DNA与蛋白质相互作用的重要工具。它可以灵敏地检测目标蛋白与特异DNA片段的结合情况，还可以用来研究组蛋白与基因表达的关系。核小体组蛋白可以发生多种翻译后的共价修饰，如乙酰化、甲基化、磷酸化、泛素化等，这些共价修饰与真核基因的表达密切相关。根据“组蛋白密码”假说，组蛋白的各种共价修 ...

(from Hogan et al. Manipulating the Mouse Embryo Cold Spring Harbor 1994) Day 1 1. Dissect embryos and place each yolk sac in a 1.5 ml microfuge tube (can be frozen at -20°C prior to extraction). ...

Use 4ml polycarbonate snap-cap tubes for all steps. Rocking of tubes should be performed at all times. All steps are carried out at room temperature unless otherwise stated. Day 0 1. Dissect embryos ...

AP Buffer: 100 mM Tris-HCl pH 9.5 100 mM NaCl 10 mM MgCl2 PTM: PBS 0.1% Tween-20 2 mM MgCl2 Since this is generally done in conjunction with lacZ staining embryo/tissue is usually fixed as per la ...

NOTE: THIS IS FINE FOR SOUTHERNS BUT NOT FOR SCREENING BY PCR. (from Ruixia 7/99 from protocol by Stef Oehen 7/94). 1. Put 1 cm tail in 1.5 ml microcentrifuge tube. 2. Add 500µl Tail Buffer a ...

These procedures were originally devised in Richard Palmiter's lab for use with tail dots (DNA spotted onto a nitrocellulose filter and probed for a transgene; quicker than doing southerns) and subseq ...

PCR screens must be designed to detect transgene DNA at the single copy level. Southern Blots analysis of transgenic mice need copy standards to estimate copy number. Copy standards are prepared by ...

This is how we test mice and rats for the presence of the transgene by PCR. It is provided for those investigators who are unfamiliar with DNA genotyping and who need to establish a genotyping method. ...

1. Obtain the last 2mm of tail and place directly into 200ul 1X PCR Buffer with Nonionic Detergents (PBND) in a 1.5ml microfuge tube. (Tails can be stored at frozen in PBS or PBND until use.) 2. Add ...

1.Cut 1/2" - 3/4" of mouse tail. (Check with your institution's Animal Studies Committee for their recommendations as to how this procedure should be implemented). 2.Add tail fragment (or t ...

1. Obtain tail biopsies from 2 to 3 week old mice: Hold mouse firmly at base of tail with one hand with the other cut off 0.5 to 1.0 cm of the tail tip with a scalpel or single edge razor blade. 2. A ...

This is how we test mice and rats for the presence of the transgene by PCR. It is provided for those investigators who are unfamiliar with DNA genotyping and who need to establish a genotyping method. ...

1. Dissect embryos and place each yolk sac in a microfuge tube (can be frozen at -20°C prior to extraction). Original method cited for mouse toes or 2 mm tail Reagents: A) 25 mM NaOH / 0.2 mM ED ...

1. Remove 0.5 cm of tail into polypropylene microfuge tube (do not mince). (The tubes must have tight-fitting caps so that there are no leaks in steps 3 and 7 below.) 2. Add 0.5 ml DNA digestion buf ...

Need 1.5-2 x 107 cells from a 2 day culture. 1. Cells are harvested as normal washed x 1 in PBS then taken up at conc. of 1.2 x 10 7 cells/ml in cold PBS. 2. Take x2 Biorad curvettes A. 0.9 ml (107 ...