TAKARA限制酶在各buffer中活性及双酶切表

• 影响限制性内切酶活性旳原因：
• DNA旳纯度 • DNA旳甲基化程度
• 酶切消化反应旳温度 • DNA旳分子构造
• 溶液中离子浓度及种类 • 缓冲液旳pH值
双酶切旳两种酶简介
• XhoⅠ酶（TaKaRa企业） 辨认序列和切割位点：
• EcoRⅠ酶（TaKaRa企业） 辨认序列和切割位点：
ቤተ መጻሕፍቲ ባይዱ
三、仪器、材料与试剂
一、试验目旳
• 经过本试验让学生掌握质粒DNA双酶切旳 原理和操作措施
二、试验原理
• 限制性内切酶是一类能辨认双链DNA分子特异性核酸序列旳DNA水解 酶。每一种内切酶能辨认DNA分子中由4～6个核苷酸构成旳特定序列
• 多种细菌能合成限制性内切酶，这是它们保护自己，降解外来DNA分 子旳主要手段。细菌细胞内旳DNA因为相应序列上旳A或C碱基旳甲 基化而不被限制性内切酶攻击，而外源DNA一旦进入细胞则立即被辨 认，双股DNA螺旋都被切断，这种现象被称为寄主控制旳限制与修饰 现象
• 水浴锅 • 移液枪和枪头 • 制冰机 • 浮板 • R-pGEX-4T-1质粒DNA • XhoⅠ酶 • EcoRⅠ酶
• 10×H Buffer
• 无菌水 • 小离心管
四、试验环节
• 质粒DNA旳双酶切 • 反应体系：
EcoRⅠ XhoⅠ 10×H Buffer 质粒DNA 无菌水 总体积
1μL 1μL 2μL 10μL～12μL≤1μg 6μL ～ 4μL 20μL（无菌水补至总体积）
• 限制性内切酶是体外剪切基因片段旳主要工具，经常与核酸聚合酶、 连接酶以及末端修饰酶等一起称为工具酶
• 限制性内切酶按限制酶旳构成、与修饰酶活性关系以及切断核酸旳情 况不同，分为三类：Ⅰ型、Ⅱ型和Ⅲ型

【原创】双酶切连接反应之全攻略（原创）双酶切连接反应之全攻略前一阵子一直在做双酶切质粒重组，失败了很多次，不过很快改善了实验方法，用2周重组了 14个质粒。

现就自己的体会，结合战友的宝贵经验，谈一下质粒重组的一些个人经验。

1、回收PCR产物：在进行PCR扩增时候，给引物两端设计好酶切位点，一般说来，限制酶的选择非常重要，尽量选择粘端酶切和那些酶切效率高的限制酶，如BamHI,HindIII,提前看好各公司的双切酶所用公用的BUFFER，以及各酶在公用BUFFER里的效率。

选好酶切位点后，在各个酶的两边加上保护碱基，其原则可参照：/upload/2006/08/13/31219184.pdf。

双酶切时间及其体系：需要强调的是很多人建议酶切过夜，其实完全没有必要，我一般酶切3个小时，其实1个小时已经足够。

应用大体系，如100微升。

纯化问题：纯化PCR产物割胶还是柱式，我推荐柱式，因为割胶手法不准，很容易割下大块的胶，影响纯化效率。

现在的柱式纯化号称可以祛除引物，既然如此，酶切掉的几个碱基肯定也会被纯化掉了。

所以，PCR产物和双酶切产物的纯化均可应用柱式纯化。

我用的是TAKARA的纯化柱试剂盒酶量的问题：以TAKARA的为例，其对1单位酶的定义如下：在50 μl 反应液中，30℃温度下反应1小时，将1 μg 的λDNA完全分解的酶量定义为1个活性单位(U)。

而该酶浓度约为15单位/微升，在除外酶降解的因素外，该酶可分解15μg的DNA，而一般从1-4ml菌液提出的 DNA约为3μg，而PCR纯化后的产物（50体系）约为3μg，所以即便全部加进去，只要纯化的质量好，酶切完全切得动。

2、酶切、回收后的PCR产物与载体的连接摩尔比的计算，很多人凭经验也可以。

但对于初学者从头认真计算则非常有必要。

回收的载体片段：回收的PCR产物片段=1：10 ，一般取前者0.03pmol，后者取0.3pmol。

pmol为单位的DNA转换为为µg单位的DNA：（X pmoles×长度bp×650）/ 1,000,000 （注：长度bp×650是该双链DNA的分子量）所得数值即为µg,也可以直接用这个公式套.1pmol 1000bp DNA=0.66μg,如载体是5380bp，则0.03pmol为0.03×5.38×0.66=0.106524µg。

前一阵子一直在做双酶切质粒重组，失败了很多次，不过很快改善了实验方法，用2周重组了 14个质粒。

现就自己的体会，谈一下质粒重组的一些个人经验。

1、回收PCR产物：在进行PCR扩增时候，给引物两端设计好酶切位点，一般说来，限制酶的选择非常重要，尽量选择粘端酶切和那些酶切效率高的限制酶，如BamHI,HindIII,提前看好各公司的双切酶所用公用的BUFFER，以及各酶在公用BUFFER里的效率。

选好酶切位点后，在各个酶的两边加上保护碱基。

双酶切时间及其体系：需要强调的是很多人建议酶切过夜，其实完全没有必要，我一般酶切3个小时，其实1个小时已经足够。

应用大体系，如100微升。

2.纯化问题：纯化PCR产物割胶还是柱式，我推荐柱式，因为割胶手法不准，很容易割下大块的胶，影响纯化效率。

现在的柱式纯化号称可以祛除引物，既然如此，酶切掉的几个碱基肯定也会被纯化掉了。

所以，PCR产物和双酶切产物的纯化均可应用柱式纯化。

我用的是TAKARA的纯化柱试剂盒3.酶量的问题：以TAKARA的为例，其对1单位酶的定义如下：在50 μl 反应液中，30℃温度下反应1小时，将1 μg 的λDNA完全分解的酶量定义为1个活性单位(U)。

而该酶浓度约为15单位/微升，在除外酶降解的因素外，该酶可分解15μg的DNA，而一般从1-4ml菌液提出的 DNA约为3μg，而PCR纯化后的产物（50体系）约为3μg，所以即便全部加进去，只要纯化的质量好，酶切完全切得动。

4.酶切、回收后的PCR产物与载体的连接:摩尔比的计算，很多人凭经验也可以。

但对于初学者从头认真计算则非常有必要。

回收的载体片段：回收的PCR产物片段=1：10 ，一般取前者0.03pmol，后者取0.3pmol。

pmol为单位的DNA转换为为µg单位的DNA：（X pmoles×长度bp×650）/ 1,000,000 （注：长度bp×650是该双链DNA的分子量）所得数值即为µg,也可以直接用这个公式套.1pmol 1000bp DNA=0.66μg,如载体是5380bp，则0.03pmol为0.03×5.38×0.66=0.106524µg。

限制性内切酶酶切的常见问题及解决方法（The common problems and solutions of restriction endonuclease digestion）The common problems and solutions of restriction endonuclease digestionRestriction enzyme digestion of the common problems and solutions, personally feel very well summed up, specially posted for you to share.Enzyme digestion problems, see enzyme instructions, the corresponding reagent company directory. The enzymes produced by different companies, the source of strains, the preparation process, purity, viability and enzymatic activity may be optimized differently,.Can be found in the above definition, unit of enzyme preservation conditions, enzyme system of [buffer and other enzymes such as buffer, double cut enzyme reaction temperature [some enzymes in 50, 55 or 30 temperature reaction, whether methylation affects the enzyme activity and whether there is an asterisk, asterisk may appear live factors and protective base, isocaudarner, isoschizomer1. enzymes can not be cut or incompleteThe 1.1 plasmid is the most common or poorly expressed inhibitor. The presence of miscellaneous proteins can affect enzyme digestion, showing A260/A280 below 1.8; inhibitors are common in phenols, salts, and ethanol; [re DNA, using reliable kits or reliable manual extraction reagents,1.2 enzyme problems: make sure the enzyme is effective [although many enzymes have expired time, but expired as long as the enzyme can be effectively cut, available. Make sure the enzyme is not effective. Mark it and replace it.[note] topic: by endonucleaseA. endonuclease, if no special requirement, keep -20~-30 degrees. Is not as low as possible, the enzyme is usually preserved in 50% glycerol buffer, when the temperature is too low, will freeze (enzyme transport process exception, because the enzyme requires low temperature transport, so the convenient situation is the use of dry ice, the enzyme will freeze, but the freezing times is limited, is a relatively better choice), if is often used, the enzyme will be repeated freezing and thawing, thereby reducing the activity. Of course, if the temperature is too high, ha ha, you want to go.B. enzyme should be used in ice box for enzyme extraction. It's clear to everyone, but it's no harm putting too much emphasis on it. Some of the students because the lab is put out, the enzyme in the ice box, but there are two ignored: when taking the enzyme, the hand does not hold the upper end of the pipe, and the grip at the bottom, equivalent to hand in to the enzyme by heating; some enzymes are placed in the ice box, but the absorption of the enzyme when will the enzyme take out. Once or twice a day, it can affect enzyme activity.C. enzyme should be tightened as soon as possible after the lid. Sometimes we can find that even if -20~-30 degrees are placed,the enzyme will freeze. That is because of personal reasons may be cut in the configuration when the enzyme reaction, enzyme tube lid open for a long time, glycerol will absorb moisture in the air, for large packaging commonly used enzyme sometimes arise freezing phenomenon. In addition, sometimes, because of careless operation, some of the broken ice congealed on the ice box fell into the enzyme tubes. (I appeared two times myself. SweatD. uptake of enzymes by tips. We used to take the enzyme tips is the smallest of the kind, suitable for 2ul and 10ul guns, but tips usually has two kinds, one is the most commonly used short tips, with it next to the enzyme, tips enzyme, the gun is almost to be inserted into the pipe, sometimes stained with enzyme in the gun body. Therefore, it may cause pollution. Then, we need to pay great attention to the action, or change to another long tips, specially for the enzyme solution.1.3 buffer problem. Some enzymes cut buffer, adding a number of easier to precipitate or dissolved components [ligase buffer is more like this], and sometimes when the enzyme buffer is not completely melted, the concentration of buffer is heterogeneous. The new buffer melts first, and the salt ion concentration is high. After a period of time, the concentration of the ions in the melt decreases. Normally, this has little effect, but problems arise if you use some enzymes that are sensitive to the concentration of ions.1.4 double enzyme buffer selection: make sure the correct buffer and reaction temperature are used [refer specifically to the enzyme buffer cut from the manufacturer and double enzymecut buffer, where NEB is availableHttp://www.neb-china.COM / CN /技术/重/ double_digests.htm1.5酶切位点的甲基化影响：有时甲基化会影响酶切，常见的有Xba I、Bcl I等。