您的位置 首页 DNA/RNA技术

如何让你的酶切反应“时半功倍”？

发布: 2016-01-15 1,669阅读 263评论

限制性核酸内切酶(以下简称限制性酶)是一类识别双链DNA中特定核苷酸序列的DNA水解酶，以内切方式水解DNA，产生5’-P和3’-OH末端。限制性酶主要分为三种类型：Ⅰ型限制酶为复合功能酶，具有限制-修饰两种功能，但在 DNA链上没有固定的切割位点，一般在离切割位点1kb到几kb的地方随机切割，不产生特异性片段。Ⅲ型酶与Ⅰ型酶基本相似，不同的是Ⅲ型酶有特异性的切割位点，但这两类酶对 DNA酶切分析的意义不大，通常所说的限制性内切酶是指Ⅱ型酶，它能够识别与切割DNA链上的特定的核苷酸顺序，产生特异性的DNA片段。

限制性酶切看似简单，但其实暗藏玄机。如果单单按照protocol上面操作，结果往往是不理想的。那么今天小编就会给你介绍一下酶切实验操作过程中需要注意的几个方面，哪怕是小细节，也至关重要。

1记得加牛血清白蛋白（Bovine Serum Albumin, BSA），达到稳定限制性酶的效果，并且减少内切酶粘附试管壁的情况。传统的方法是单独购买BSA溶液，现在越来越多的缓冲液例如Tango Buffer或是Mastermix都含有BSA，从而使酶切实验更为简便。

 

2如果限制性酶的反应效率在含盐缓冲液中(e.g., NEBuffer 3.1) 很低，说明很有可能这种酶对盐量敏感。在DNA 的制备过程中，要尽量清除掉溶液中的盐分。

 

3限制性酶的总体积要少于总反应液体积的10%。用于保存酶的甘油如果过多，会抑制酶切反应，大大降低酶切的效率。

 

4在保证第3条的前提下，加尽可能多的限制性酶来提升反应效率。这里要强调下一单位（1U）的定义，一单位酶可以在一小时内标准反应条件下酶切1 µg的DNA。如果担心甘油会抑制酶切，可以考虑购买单位浓度比较高的限制性酶。每一个生产商都会标明浓度单位，例如10U/µL。

 

5进行无酶切、单酶切测试。不管是什么实验，都要有控制实验来证明结果的有效性。不加入任何限制性酶，或是加入单一酶都可以很好的验证限制性酶的效率并且分辨出假阳性实验结果。

 

6查看限制性酶的反应效率表和相对应的保护碱基。限制性酶除了识别特定的DNA序列外，酶蛋白还要占据识别位点两边的若干个碱基，这些碱基对内切酶稳定的结合到DNA双链并发挥切割DNA作用是有很大影响的。每一个酶的工作效率和保护碱基的长短不尽相同，要根据个人使用的酶的特点来设计实验才能达到理想的效果。

 

以下链接是保护碱基数目和酶切活性对应值表，供小伙伴们参考。

http://wenku.baidu.com/view/312a400ff12d2af90242e6d3.html

https://www.neb.com/~/media/NebUs/Files/Chart%20image/cleavage_olignucleotides_old.pdf

以上技巧是小编根据个人经验和网上资料整理得来的，如果你有更好的建议和意见，也欢迎各位小伙伴来信告诉我们。

图片来自网络，内容由BioEngX原创，转载请注明！

1.25K赞
标签:



相关文章

回复 porno dla dzieci 取消回复

您的电子邮箱地址不会被公开。 必填项已用*标注

评论列表(263)

  10. Эта статья предлагает живое освещение актуальной темы с множеством интересных фактов. Мы рассмотрим ключевые моменты, которые делают данную тему важной и актуальной. Подготовьтесь к насыщенному путешествию по неизвестным аспектам и узнайте больше о значимых событиях.
    Исследовать вопрос подробнее – https://vyvod-iz-zapoya-1.ru/

    回复

  31. Do you have a spam problem on this website? I also am a blogger, and I was wondering your situation; we have developed some nice practices and we are looking to exchange techniques with other folks, be sure to shoot me an email if interested.

    回复

  32. Well I definitely enjoyed reading it. This subject offered by you is very constructive for correct planning.

    回复

  36. Wow! This blog looks exactly like my old one! It’s on a totally different subject but it has pretty much the same page layout and design. Excellent choice of colors!

    [url=https://entexpert.in/official-india-fun88-app-seamless-betting-experience/]entexpert.in[/url]

    回复

  62. His son bucks on the lid as he turns beet red from his carnal machinations.

    回复

  69. “Back in the day, back before we turned into a modern society, a man would grab another man’s balls and swear an oath on them in his hand and state his words to be true. To be a friend” His father says. “Do you know what I mean, son?”

    回复

  76. His father rakes the clear juice of his son’s leaking manhood over the boy’s tensed cockhead. “Then show me, father. Show me what it means, TO BE A MAN?” He says with an excitement building in his voice.

    回复

  78. “Maybe his dad is right.” He thinks to himself as he continues with his intense fondling of his fiery red cock. “I figured you did.” His father says. “You play on a team, so you know the purpose of teamwork.”

    回复

  79. Nicely put. With thanks.
    casino sverige online [url=https://usagamblinghub.com/#]play casino online[/url] pointsbet online casino pa

    回复

  80. You made your position pretty nicely!.
    casino online debit card [url=https://usacasinomaster.com/#]online casinos usa[/url] online casino no deposits

    回复

  81. Whoa a lot of awesome advice.
    online casino biggest win [url=https://luckyusaplay.com/#]best online casino reddit no deposit bonus[/url] casino online espaГ±a tragamonedas

    回复

  82. Awesome posts. Kudos.
    liberty online casino [url=https://luckyusaplay.com/#]games casino free online[/url] software para casinos online gratis

    回复

  88. Nicely put, Thanks!
    best maine online casino site [url=https://usaplayerscasino.com/#]best online casinos for usa[/url] free casino slots games online com no downloads

    回复

  89. Lovely forum posts. Thank you.
    sa best online casino [url=https://usagamblinghub.com/#]new online casino usa ok[/url] credit cards online casino

    回复

  91. You said it nicely.!
    legit online casino games that pay real money [url=https://usacasinomaster.com/#]casino free games online[/url] besst online casino united states

    回复

  93. Cheers. Excellent stuff.
    cashtocode online casino [url=https://luckyusaplay.com/#]casinos gambling online[/url] online casinos mastercard

    回复

  95. Many thanks. Ample facts.
    online casino real money free bonus [url=https://uscasinoguides.com/#]newest online casinos usa[/url] legit casino online

    回复

  97. You revealed that really well!
    mines casino online [url=https://usaplayerscasino.com/#]online casino bonus[/url] online casino evolution gaming

    回复

  99. Well spoken indeed. !
    top online casino deals [url=https://usagamblinghub.com/#]online usa casinos[/url] online casinos that accept visa debit

    回复

  100. You reported this well.
    free bonus codes online casino [url=https://usacasinomaster.com/#]best online casino reddit no deposit bonus[/url] silversands online casino

    回复

  102. Cheers! An abundance of material!
    www casino online com gratis [url=https://luckyusaplay.com/#]casino games online free[/url] free no deposit bonus codes for online casinos

    回复

  106. Cheers. I like it.
    free online casino no deposit promo codes [url=https://uscasinoguides.com/#]no deposit online casinos[/url] online casino debit card withdrawal

    回复

  107. You actually reported it exceptionally well!
    best vpn for online casino [url=https://usaplayerscasino.com/#]casino online no deposit[/url] 2021 online casino bonus codes

    回复

  109. Kudos. Ample write ups.
    online casinos in virginia [url=https://usacasinomaster.com/#]most trusted online casinos for usa players[/url] online casino bonus michigan

    回复

  111. Perfectly expressed really. !
    best online casino uae [url=https://luckyusaplay.com/#]no deposit online casino[/url] ballys online casino reviews

    回复

  114. Factor nicely considered.!
    play free casino games online for free [url=https://uscasinoguides.com/#]online games casino[/url] west virginia online casino no deposit bonus

    回复

  116. Thanks a lot, Ample stuff.
    online casino advantage play [url=https://usagamblinghub.com/#]biggest online casino[/url] casino online free money no deposit

    回复

  118. Nicely put. Appreciate it.
    casino america online [url=https://usacasinomaster.com/#]new online casinos usa[/url] leovegas casino online

    回复

  123. Nicely put, Thanks a lot.
    online casinos that accept american express [url=https://uscasinoguides.com/#]best online casinos[/url] play real money online casino

    回复

  124. Many thanks! Lots of posts.
    online casino for usa [url=https://usaplayerscasino.com/#]no deposit online casinos[/url] south african online casinos with no deposit coupons

    回复

  126. You stated that effectively.
    casinos online con bonos sin depГіsito [url=https://usagamblinghub.com/#]online casino free bonus[/url] online casino bitcoin withdrawal

    回复

  127. Nicely put. Regards.
    huge jackpot in casino online 2022 [url=https://usacasinomaster.com/#]online casinos[/url] online casino app in india

    回复

  128. Reliable tips. Regards.
    10$ deposit online casino [url=https://luckyusaplay.com/#]casino live online[/url] casino online juego del dinosaurio

    回复

  131. With thanks! Great stuff!
    888 casino online blackjack [url=https://uscasinoguides.com/#]free casino online games[/url] free online casino australia

    回复

  133. Appreciate it, An abundance of facts!
    online casino tipico [url=https://usagamblinghub.com/#]casino free games online[/url] most popular online casino nz

    回复

  134. This is nicely said. .
    juwa casino online 777 guia downloadable content apk [url=https://usacasinomaster.com/#]casino online games free[/url] 1xbet online casino review

    回复

  136. Kudos! Lots of knowledge!
    wow slots: vip casino online [url=https://luckyusaplay.com/#]best online casino welcome bonus no deposit[/url] australian online live casino

    回复

  138. You actually revealed that superbly!
    online casino content [url=https://uscasinoguides.com/#]online live casino[/url] habanero online casino

    回复

  139. Seriously quite a lot of helpful knowledge.
    online casino paysafecard bezahlen [url=https://usaplayerscasino.com/#]casino online free[/url] casino online fuera de espaГ±a

    回复

  141. Thank you! Quite a lot of tips.
    best indian online casino [url=https://usagamblinghub.com/#]online casino free spins no deposit[/url] online casino mit freispielen bei anmeldung

    回复

  144. You said it wonderfully.
    caesars casino online free slots [url=https://luckyusaplay.com/#]american casino online[/url] el dino casino online

    回复

  146. Nicely put. Kudos.
    zodiac casino online spielen [url=https://uscasinoguides.com/#]online casino paypal[/url] texas holdem online casino

    回复

  147. Incredible all kinds of terrific info.
    online casino no deposit bonus free spins australia [url=https://usaplayerscasino.com/#]online casino paypal[/url] banking online casino

    回复

  149. Beneficial tips. With thanks!
    payid casino online [url=https://usagamblinghub.com/#]casino live online[/url] gta 5 online casino heist scope all p.o.i

    回复

  151. Terrific data, Cheers!
    kostenlose online casino spiele [url=https://usacasinomaster.com/#]online casino usa[/url] casino wars online

    回复

  152. Nicely put, Thanks!
    play casino table games online [url=https://luckyusaplay.com/#]social casinos online[/url] best casino online uk

    回复

  198. I enjoyed reading this article. Its thought-provoking and well-presented. Lets discuss this further. Check out my profile!

    回复
联系我们

联系我们

(44)07934433023

在线咨询: QQ交谈

邮箱: info@bioengx.org

关注微信
微信扫一扫关注我们

微信扫一扫关注我们

关注微博
返回顶部