細(xì)菌轉(zhuǎn)化實(shí)驗(yàn)英漢兩種實(shí)驗(yàn)方案

1、一實(shí)驗(yàn)?zāi)康?以pGLO質(zhì)粒轉(zhuǎn)化大腸桿菌為例，學(xué)習(xí)轉(zhuǎn)化的基本原理及方法。2驗(yàn)證DNA是遺傳物質(zhì)，加深對(duì)中心法則的理解。二實(shí)驗(yàn)原理轉(zhuǎn)化（transformation）是指一段同源或異源的DNA轉(zhuǎn)入受體細(xì)胞并得到表達(dá)的水平基因的轉(zhuǎn)移過程，是現(xiàn)代分子生物學(xué)研究和基因工程不可缺少的重要技術(shù)。目前常用的轉(zhuǎn)化方法有CaCl2法(化學(xué)轉(zhuǎn)化法)和電轉(zhuǎn)化法。本實(shí)驗(yàn)是用pGLO細(xì)菌轉(zhuǎn)化試劑盒中提供的pGLO質(zhì)粒來轉(zhuǎn)化大腸桿菌，所用方法為CaCl2轉(zhuǎn)化法。 pGLO質(zhì)粒： pGLO質(zhì)粒上主要含有兩個(gè)基因，一個(gè)為編碼綠色熒光蛋白(GFP)的基因，一個(gè)為抗生素氨芐青霉素抗性基因。此外，該質(zhì)粒還整合有一個(gè)特殊的參與受體細(xì)胞

2、中綠色熒光蛋白表達(dá)的基因調(diào)控體系，轉(zhuǎn)化細(xì)胞中的綠色熒光蛋白基因只有在培養(yǎng)基中存在阿拉伯糖時(shí)才能啟動(dòng)表達(dá)。轉(zhuǎn)化子細(xì)胞將在不含阿拉伯糖的培養(yǎng)基上呈白色而在含阿拉伯糖的培養(yǎng)基上顯綠色熒光，這種熒光在長(zhǎng)波紫外燈下即可觀察到。 三實(shí)驗(yàn)材料 受體菌：E.coli K12 HB101質(zhì)粒：pGLO plasmid轉(zhuǎn)化液（CaCl2）氨芐青霉素（amp）阿拉伯糖（ara）培養(yǎng)基：固體LB、液體LB接種環(huán)、移液器等四實(shí)驗(yàn)步驟 1. 準(zhǔn)備平板： 每組：1塊LB平板，2塊LB/amp平板，1塊LB/amp/ara平板 2. 準(zhǔn)備感受態(tài)細(xì)胞：用250µl無菌水或轉(zhuǎn)化液懸浮試劑盒中提供的大腸桿菌菌粉，此即感受

3、態(tài)細(xì)胞。 3. 活化受體細(xì)胞：挑取1環(huán)E.coli K12 HB101菌液，于LB培養(yǎng)基上37活化16-24h。 4. 轉(zhuǎn)化：1）在兩只無菌離心管上分別標(biāo) 記 DNA, -DNA。 2）在上述兩管中分別加入 250µl轉(zhuǎn)化液。 3) 迅速置于冰上。 4）各挑取活化好的受體菌的一 個(gè)單菌落，懸浮于兩管轉(zhuǎn)化液中。 5）在標(biāo)有 DNA的管中加入一 環(huán)質(zhì)粒DNA，而標(biāo)有-DNA的管中不加。 6）將上述兩管于冰上放置10min。 7）在準(zhǔn)備好的培養(yǎng)基底部如左圖。8）兩只小管放入42水浴中處理50秒，再迅速放于冰上2min。 9）在兩只小管中分別加入250µl LB-肉湯。 10）從

4、DNA小管中分別吸取100µl滴加在兩個(gè)標(biāo)有 DNA的平板上，從-DNA小管中分別吸取100µl滴加在兩個(gè)標(biāo)有-DNA的平板上。11）用無菌接種環(huán)將滴加的液體均勻涂布在平板上。12）將上述四只平板固定，并于42培養(yǎng)箱中培養(yǎng)2天。 13）在長(zhǎng)波紫外燈下觀察結(jié)果，記錄各平板上的菌落數(shù)Bacterial Transformation(細(xì)菌轉(zhuǎn)化) 作者：佚名 來源：ucc 時(shí)間：2008-5-18 IntroductionTransformation is a technique to introduce DNA into bacterial cells. There a

5、re 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 Ð do not use

6、an excessive amount of DNA, both in terms of concentration and actual volume (less than 1 µg and less than 10 µl). Note,protein (e.g. Ligase) will reduce transformation efficiency, but it is not always necessary to remove prior to transformation.B Heat shock Ð necessary for DNA uptake

7、. Time heat shock carefully Ð excessive heat shock will kill the bacteria and the transformation will fail.C Recovery Ð 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 Select

8、ion Ð 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 Ð always thaw slowly on ice and do not hold

9、the base of the eppendorf tube. The compency of the bacteria is also important-“sub-cloning efficiency” means about 106 colonies are produced per µg of (purified) DNA. “Library efficiency” can mean in excess of 109 colonies produced per µg of (purified) DNA. For subcloning and mutagenesis

10、is normally sufficient, although “Library efficiency” bacteria may be useful if problems arise.Materials requiredCompetent cells       nutrient brothDNA                   

11、0;    Antibiotic platesPlate spreader           Bunsen burnerL-broth agar plates   Ethanol CautionBunsen burners have a naked flames Ð do not leave unattended.Three transformations must be performed: Experiment

12、al reaction(s) Plasmid or positive control Ð this shows that E. coli are competent for DNA uptake H2O or negative control Ð this shows that there are no false positivesBacterial Transformation1. Add 1-10 µl of the DNA (Experimental reaction or positive/negative control) to a

13、 vial(20-200 µl) 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 42ûC without shaking (time varies by strain).4. Immediately transfer the tubes to ice and incubate for 2 min.5. Add 50-500

14、81;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 µl from each transformation on a L-broth agar plates containing antibiotics at the appropriate concentration. Incubate plates for 5-10 mins at room temperature, the

15、n 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.細(xì)菌轉(zhuǎn)化 transformation BIOX.CN 2005-7-11 20:49:11來源：生命經(jīng)緯 細(xì)菌的融合的一種形態(tài)，亦即某一菌株（供體菌）的一部分遺傳性狀移到另一菌株（變體菌）的一種遺傳雜交形態(tài)。轉(zhuǎn)化是指外源DNA，即從