DNA实验技术

将外源基因通过体外重组后导入受体细胞，使该基因能在受体细胞内复制、转录、翻译和表达，整个操作称为基因重组技术。要实施该技术必须具备四大要素：工具酶、载体、基因和受体（宿主）细胞。

质粒DNA的提纯是基因工程实验中最常用的试验方法之一，质粒DNA提纯效率和质量与其后续实验步骤（如PCR扩增、酶切、连接、转化大肠杆菌和转染真核细胞等）的成功与否有着直接的关系，因此高效快速地从细菌细胞中提纯质粒DNA 具有重要的意义，本文主要介绍了由QIAGEN plasmid Midi试剂盒方法提取质粒的方法。

掌握DNA重组技术的基本原理和基本方法和熟悉质粒DNA的小量制备及酶切鉴定方法

UV shadowing is a technique for visualizing nucleic acids separated on acrylamide/urea gels. The technique utilizes shortwave UV light (254 nm) and a fluor-coated TLC plate. We recommend UV shadowing ...

Restriction enzyme digestions are performed by incubating double-stranded DNA molecules with an appropriate amount of restriction enzyme in its respective buffer as recommended by the supplier and at ...

Restriction enzymes are expensive ($40 to $200 a vial): keep the enzymes at -20℃. Plan your digests to be small and convenient. The digest is composed of DNA sample (volume should not be more than abo ...

This is a question asked quite a bit most recently (late September 1995) in the newsgroup. Here are two methods and who submitted them. ...

本方法的核心部分是医科院基础所的生化脂蛋白组的吴刚老师所创，我在其基础上进行了一些改动，主要是DNA回收上采取了更简单的方法。（本方法最适用于双酶切制作片断并进行克隆 的情况。对于分步酶切制作片断，也可以使用本方法，但需要加倍起始酶切DNA的量。）

(一) 鼠肝DNA的制备

I usually use Linear polyacrylamide as a carrier for DNA precipitation by EtOH. I have read about using glycogen as a carrier I would like to know if anyone knows a method avoiding not to use acrylami ...

真核细胞的mRNA在加工过程中有一个比喻为“穿鞋戴帽”的过程，因此mRNA的3'末端都带有一段Poly A，这是利用逆转录酶制备cDNA文库的基础。但是由于cDNA的5'端的序列各不相同，如何获得全长的cDNA，如何扩增由微量的mRNA逆转录得到的cDNA文库、如何利用已知片断序列得到全长的cDNA（即RACE），曾经是一个令人困扰的问题。

I have read some books but cannot find the answer.I think NaAc may increase the ionic strength to stabilize DNA duplexes.Is it right?

差异基因表达的研究受到了广泛的关注，常用的技术有DD-PCR；GENE-FISHING；GENE CHIP等。简单介绍如下： DDRT―PCR技术即mRNA差异显示聚合酶链式反应技术，此技术是以PCR技术和聚丙烯凝胶电泳技术为基础，结合银染或放射性自显影等显色技术，能快速有效地鉴定并克隆两个或多个平行材料之间的差异表达基因。DDRT―PCR技术的基本过程如下：①提取两组平行材料中的总RNA或m ...

Plasmid isolated by this procedure can be used routinely for electrophoretic analysis restriction endonuclease digestion and transformation of E. Coli. sequencing PCR and most other molecular biologic ...

Identify appropriate celltype over-expressing corresponding gene.Find out if transcription can be stimulated further eg by induction (note: the higher the mRNA level of the gene in question the easier ...

The gel shift or electrophoretic mobility shift assay provides a simple and rapid method for detecting DNA-binding proteins. This method has been used widely in the study of sequence-specific DNA-bind ...

1) The ligation mixture contains the following: vector DNA (~100 ng) insert DNA (equimolar or 2 or 3 X molar concentration of vector) water added to 18 µl 2) Heat the mixture at 45 °C for 5 min. to me ...

Prepare a ligation mix:Ligation Mix (2x)10x ligase buffer1.0 mldigested vector(0.1 mg/ml)1.0 mlH2O6.0 mltotal8.0 mldivide ligation mix into two Eppendorf tubesLigation Rxn InsertControlligation mix ...

Prepare a 100 µl reaction mixture containing the DNA of interest in the appropriate 1X restriction enzyme buffer. Divide the reaction mixture between five prechilled microcentrifuge tubes such that tu ...

Gel Shift or Band Shift Assay or Electrophoretic Mobility Shift Assay (EMSA) is a technique for studying gene regulation and determining protein:DNA interactions. The assay is based on the observation ...