基因克隆

Introduction This manual is a compilation of many of the everyday methods used in the average molecular biology laboratory with emphasis on the techniques for large scale DNA sequencing protocols and ...

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、片断平移的克隆 （也适用于多片断连接） 简介：将用作载体的质粒A（制作大片断）酶切3μL ，要切出小片断的质粒B酶切7μL；琼脂糖 ...

PCR产物克隆大致分为两类，即平头连接和粘头连接。 平头连接是将制备好的平头载体和补平或削平的PCR 产物直接进行连接。载体可用EcoR V或Sma I切成平头；PCR 产物纯化后，可以在22℃用DNA聚合酶I作用30min（利用该酶所具有的3’→5’外切酶活性和5’→3’的聚合酶活性）。如果要求不高，PCR 产物也可不加处理。如 ...

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The pGL3 Luciferase Reporter Vectors provide a basis for the quantitative analysis of factors that potentially regulate mammalian gene expression. These factors may be cis-acting, such as promoters and enhancers, or trans-acting, such as various DNA-binding factors. The backbone of t ...

TA Cloning exploits the terminal transferase activity of some DNA polymerases such as Taq polymerase. This enzyme adds a single, 3'-A overhang to each end of the PCR product. This makes it possible to clone this PCR product directly into a linearized cloning vector with single, 3'-T overhangs. The PCR products with dA overhang, are mixed with this vector in high proportion. The complementary overhangs of "T" vector and PCR product will be ligated under the action of T4 DNA ligase.

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ALKALINE PHOSPATASE(CIP) DIGESTION Digestion of protruding 5'-ends 1. Precipitate digested DNA and resuspend in a 100ml of 1X CIP Buffer. 2. Add 1U CIP for 100pmol 5'-ends. 2g linearized 5kb plasmid Å 1.4pmol 5'-ends 3. Incubate 30' @ 37℃. 4. Add 1ml 0.5M EDTA (to 5mM). 5. Phenol-Sevag extract to remove all the enzyme and NaOAc/EtOH ppt. 6. Resuspend in ddH2O or TE-4 @ Å100ng/l. Calf Intestinal Alkaline Phosphatase Boehringer Mannhein #713 023 0.1M Tris-HCl, pH 8.5 10mM ZnCl2 10mM MgCl2 Digestio