细菌转化实验英汉两种实验方案.doc

一实验目的1以pGLO质粒转化大肠杆菌为例，学习转化的基本原理及方法。2验证DNA是遗传物质，加深对中心法则的理解。二实验原理转化（transformation）是指一段同源或异源的DNA转入受体细胞并得到表达的水平基因的转移过程，是现代分子生物学研究和基因工程不可缺少的重要技术。目前常用的转化方法有CaCl2法(化学转化法)和电转化法。本实验是用pGLO细菌转化试剂盒中提供的pGLO质粒来转化大肠杆菌，所用方法为CaCl2转化法。 pGLO质粒： pGLO质粒上主要含有两个基因，一个为编码绿色荧光蛋白(GFP)的基因，一个为抗生素氨苄青霉素抗性基因。此外，该质粒还整合有一个特殊的参与受体细胞中绿色荧光蛋白表达的基因调控体系，转化细胞中的绿色荧光蛋白基因只有在培养基中存在阿拉伯糖时才能启动表达。转化子细胞将在不含阿拉伯糖的培养基上呈白色而在含阿拉伯糖的培养基上显绿色荧光，这种荧光在长波紫外灯下即可观察到。 三实验材料 受体菌：E.coli K12 HB101质粒：pGLO plasmid转化液（CaCl2）氨苄青霉素（amp）阿拉伯糖（ara）培养基：固体LB、液体LB接种环、移液器等四实验步骤 1. 准备平板： 每组：1块LB平板，2块LB/amp平板，1块LB/amp/ara平板 2. 准备感受态细胞：用250l无菌水或转化液悬浮试剂盒中提供的大肠杆菌菌粉，此即感受态细胞。 3. 活化受体细胞：挑取1环E.coli K12 HB101菌液，于LB培养基上37活化16-24h。 4. 转化：1）在两只无菌离心管上分别标 记 DNA, -DNA。 2）在上述两管中分别加入 250l转化液。 3) 迅速置于冰上。 4）各挑取活化好的受体菌的一 个单菌落，悬浮于两管转化液中。 5）在标有 DNA的管中加入一 环质粒DNA，而标有-DNA的管中不加。 6）将上述两管于冰上放置10min。 7）在准备好的培养基底部如左图。8）两只小管放入42水浴中处理50秒，再迅速放于冰上2min。 9）在两只小管中分别加入250l LB-肉汤。 10）从 DNA小管中分别吸取100l滴加在两个标有 DNA的平板上，从-DNA小管中分别吸取100l滴加在两个标有-DNA的平板上。11）用无菌接种环将滴加的液体均匀涂布在平板上。12）将上述四只平板固定，并于42培养箱中培养2天。 13）在长波紫外灯下观察结果，记录各平板上的菌落数BacterialTransformation(细菌转化) 作者：佚名 来源：ucc 时间：2008-5-18 IntroductionTransformation is a technique to introduce DNA into bacterial cells. There are many variations on a common theme, but the key points are listed below. Check details with supplier of competent bacteria and note that variations in timings and volumes will vary with application and bacterial strain.There are four stages:A Mix DNA/bacteria and incubate on ice do not use an excessive amount of DNA, both in terms of concentration and actual volume (less than 1 g and less than 10 l). Note,protein (e.g. Ligase) will reduce transformation efficiency, but it is not always necessary to remove prior to transformation.B Heat shock necessary for DNA uptake. Time heat shock carefully excessive heat shock will kill the bacteria and the transformation will fail.C Recovery prior to selecting for transformed bacteria with antibiotics, it is necessary to allow them to recover in rich medium (e.g. LB, SOC or 2YT) for 30-60 mins at 37 .D Selection essential to isolate (as single colonies) the bacteria which have taken up DNA.This is usually performed on solid medium (LB-agar) in the presence of antibiotics. Cells are incubated at 37 overnight.Competent bacteria are extremely fragile always thaw slowly on ice and do not hold the base of the eppendorf tube. The compency of the bacteria is also important-“sub-cloning efficiency” means about 106 colonies are produced per g of (purified) DNA. “Library efficiency” can mean in excess of 109 colonies produced per g of (purified) DNA. For subcloning and mutagenesis is normally sufficient, although “Library efficiency” bacteria may be useful if problems arise.Materials requiredCompetent cells nutrient brothDNA Antibiotic platesPlate spreader Bunsen burnerL-broth agar plates EthanolCautionBunsen burners have a naked flames do not leave unattended.Three transformations must be performed:Experimental reaction(s)Plasmid or positive control this shows that E. coli are competent for DNA uptakeH2O or negative control this shows that there are no false positivesBacterial Transformation1. Add 1-10 l of the DNA (Experimental reaction or positive/negative control) to a vial(20-200 l) of competent E. coli cells and mix gently. Do not mix by pipetting up and down.2. Incubate on ice for 30 min.3. Heat shock the cells for 30 sec at 42C without shaking (time varies by strain).4. Immediately transfer the tubes to ice and incubate for 2 min.5. Add 50-500 l nutrient broth (room temperature).6. Cap the tube tightly and shake the tubes at 37 for 30 min. Place on ice.7. Spread 50-500 l from each transformation on a L-broth agar plates containing antibiotics at the appropriate concentration. Incubate plates for 5-10 mins at room temperature, then invert the plates and incubate overnight at 37.Most strains require 12-18 hours to form colonies. Do not incubate for excessive times as satelite colonies will form. Plates/colonies can be stored for a few days at 4 if not to be used immediately.AppendixGibco, New England Biolabs and Stratagene have comprehensive guides to transformation.细菌转化 transformation BIOX.CN 2005-7-11 20:49:11来源：生命经纬 细菌的融合的一种形态，亦即某一菌株（供体菌）的一部分遗传性状移到另一菌株（变体菌）的一种遗传杂交形态。转化是指外源DNA，即从供体菌抽提出来