您的位置 首页 DNA/RNA技术

QuikChange定点突变方法简说

定点突变技术是指通过聚合酶链式反应(PCR)等方法对DNA片段中特定位置碱基进行替换,删除或添加新的碱基的技术。体外定点突变技术是当前生物,医学各领域研究中的一种重要的实验手段,能够用来探究蛋白质结构与功能之间的关系,优化基因表达,改造载体等目的。小编今天为大家介绍一种常用的体外定点突变的方法,QuikChange Site-directed Mutagenesis。

4

QuikChange方法以质粒作为模板,经过PCR反应,Dpn I 酶切和转化三个步骤,能够在一天内完成定点突变实验。当然在实验以前,我们需要设计包含有目的突变点的引物。下面小编将简单介绍引物设计,PCR反应,Dpn I 酶切以及转化这几个步骤。详细内容可参考Agilent QuikChange II XL Site-Directed Mutagenesis Kit 手册。

引物设计

引物应该是一对互补的寡核苷酸链,其中含有目的突变位点。引物长度在25-45碱基之间, 引物的Tm值要大于78 oC,长于45个碱基的引物将有可能形成引物二聚体,从而影响突变反应的效率。目的突变点应该位于引物的中间位置,突变位点两侧各有10-15个与模板一致碱基。另外引物的GC含量至少应为40%, 并且有至少一个G或C碱基位于末端。小编推荐利用Agilent官网中的软件来设计引物,www.agilent.com/genomics/qcpd

PCR 反应

PCR反应需要考虑两个方面:一个是PCR反应体系,另一个是PCR反应条件。在设计PCR反应体系时,需要注意的是引物的浓度。反应体系中引物需要过量,通常一个50 μL的反应体系内应该含有超过125g的引物。在首次尝试设计反应体系时,可以加入10 ng的质粒模板,如果反应没有成功,需要优化模板的用量,范围应该在5 ng-50 ng之间。设计PCR反应条件时,常选择5 min的延伸时间,18个循环。循环数大于18, 将有可能引入非特异突变。

Dpn I 酶切

Dpn I限制性内切酶用来降解甲基化的模板DNA以避免后续转化可能导致的假阳性克隆。通常情况下,50 μL反应体系在中加入1 μL Dpn I酶,37 oC放置1 h 即可消除模板DNA。

转化

Quikchange方法的好处是不需要进行再次克隆,可以直接将Dpn I酶切的产物转化进入大肠杆菌的感受态细胞中。转化采用常规的热激方法即可。转化后平板需要在培养箱内放置不少于16 h。最后挑选阳性克隆,测序进行验证。在实验过程中,可能会遇到各种问题,比如转化效率很低,突变效率不高,出现假阳性克隆等等,详细解决对策可点击本链接

图片来自网络,内容由BioEngX原创,转载请注明!


扫描下方二维码关注BioEngX官方微信公众平台
qrcode_for_gh_1d4074a25cf9_258

作者: 于浩然

本科及硕士毕业于天津大学,博士毕业于伦敦大学学院。现就职于浙江大学化学工程与生物工程学院,PI,博导。研究方向为蛋白质工程、生物催化剂、合成生物学等。



发表回复

您的电子邮箱地址不会被公开。 必填项已用*标注

评论列表(193)

  1. I really love your blog.. Pleasant colors & theme. Did you develop this website yourself? Please reply back as I’m attempting to create my own personal blog and would love to know where you got this from or exactly what the theme is called. Cheers!

    https://supplier.mercedes-benz.com/external-link.jspa?url=https://canvas.instructure.com/eportfolios/3012957/emilianomcpd085/What_I_Wish_I_Knew_a_Year_Ago_About_Japan_travel_planning {japan travel|travel japan|japan travel guide|esim japan travel|travel insurance japan|travel to japan|japan travel sim|japan travel app|japan web travel|japan official travel app|japan travel blog|japan travel insurance|japan travel web|travel japan website|esim for japan travel|travel insurance in japan for foreigners|travel japan web|japan travel navitime|japan travel planner|rakuten travel japan|japan travel by navitime|ptt japan travel|e travel japan registration|japan travel agency|navitime japan travel|travel in japan|travel quotes japan|travel agency in japan|caption for japan travel|japan travel sim card|cute japan travel quotes|travel requirements from japan to philippines 2023|travel agency in japan for foreigners|sim card japan travel|japan travel tips|japan quotes travel|japan travel sim 使えない|japan travel qr code|bcd travel japan|e travel japan|e travel japan to philippines|japan e travel|japan travel recommend|japan travel quotes|japan travel kk|japan travel ptt|japan travel website|japan travel esim|e travel pass japan|travel sim japan|solo travel japan|e travel registration japan|travel japan wifi|japan wonder travel|travel agency japan|japan travel requirements 2023|travel itinerary sample japan|japan travel map|japan travel card|travel sim card in japan|travel japan qr code|travel japan insurance|japan travel sim 日本人|japan to philippines travel requirements|e travel qr code japan|luxury travel japan|japan hands free travel|japan travel english|japan travel restrictions|japan travel plan|e travel pass japan requirements|navitime for japan travel|travel itinerary japan|japan travel itinerary|luggage free travel japan|verizon travel pass japan|travel adapter japan|hands free travel japan|travel to philippines from japan requirements|travel to australia from japan|japan travel pass|japan travel captions|japan travel kyoto|japan hands-free travel|travel insurance japan to europe|travel insurance from japan|travel sim card japan|japan travel news|travel insurance for japan|travel esim japan|travel

  2. An outstanding share! I have just forwarded this onto a colleague who has been conducting a little homework on this. And he actually bought me breakfast due to the fact that I found it for him… lol. So allow me to reword this…. Thanks for the meal!! But yeah, thanx for spending the time to talk about this matter here on your web page.

    https://www.gosnursesleague.org/

  3. I really love your website.. Excellent colors & theme. Did you develop this website yourself? Please reply back as I’m trying to create my own site and would like to know where you got this from or what the theme is called. Thanks!

    http://www.lasvegastourgirls.com/

  4. That is a really good tip particularly to those fresh to the blogosphere. Simple but very precise info… Many thanks for sharing this one. A must read post!

    https://iptvmade.com

  5. Hi there! I could have sworn I’ve been to this web site before but after looking at some of the articles I realized it’s new to me. Regardless, I’m certainly happy I found it and I’ll be book-marking it and checking back often!

    https://rent2ownsmart.com

  6. I seriously love your blog.. Excellent colors & theme. Did you create this website yourself? Please reply back as I’m looking to create my very own blog and would like to learn where you got this from or exactly what the theme is called. Thanks!

    https://www.hishypesports.com

  7. I would like to thank you for the efforts you have put in penning this website. I really hope to see the same high-grade blog posts from you in the future as well. In fact, your creative writing abilities has encouraged me to get my own blog now 😉

    https://iwin68.bio/

  8. Hi there, I do believe your website could possibly be having browser compatibility problems. Whenever I take a look at your blog in Safari, it looks fine however, if opening in IE, it has some overlapping issues. I just wanted to give you a quick heads up! Aside from that, wonderful website!

    https://iklimbantendki.id/news/

  9. I blog frequently and I really thank you for your content. This great article has truly peaked my interest. I’m going to take a note of your blog and keep checking for new details about once a week. I subscribed to your RSS feed too.

    https://www.funkyrepublicvapes.org

  10. Great site you have got here.. It’s difficult to find quality writing like yours nowadays. I seriously appreciate individuals like you! Take care!!

    https://www.jimjeans.com/

  11. Hello, I think your site might be having browser compatibility issues. When I look at your website in Safari, it looks fine but when opening in Internet Explorer, it has some overlapping. I just wanted to give you a quick heads up! Other then that, very good blog!

    https://knowyourmeme.com/users/fogpasta3/

  12. Hiya, I’m really glad I have found this information. Nowadays bloggers publish only about gossips and net and this is actually annoying. A good website with interesting content, that’s what I need. Thanks for keeping this web-site, I will be visiting it. Do you do newsletters? Can’t find it.

    http://atlas.dustforce.com/user/pestcontrol085

  13. After I initially left a comment I seem to have clicked the -Notify me when new comments are added- checkbox and from now on whenever a comment is added I get four emails with the exact same comment. Perhaps there is a way you can remove me from that service? Cheers.

    https://casino5588.com/

  14. I was wondering if you ever considered changing the page layout of your site? Its very well written; I love what youve got to say. But maybe you could a little more in the way of content so people could connect with it better. Youve got an awful lot of text for only having 1 or two images. Maybe you could space it out better?

    https://pixelfactory.es/desarrollo-web-bilbao/

  15. Pingback: Cocuk pornosu
  16. Pingback: porn
  17. FitSpresso: What Is It?FitSpresso is a natural weight loss aid designed for individuals dealing with stubborn weight gain. It is made using only science-backed natural ingredients.

    https://youtu.be/3d0xCIymv_o

  18. Some tips i have seen in terms of pc memory is that often there are technical specs such as SDRAM, DDR and so forth, that must match up the specifications of the mother board. If the personal computer’s motherboard is reasonably current while there are no main system issues, replacing the memory literally will take under sixty minutes. It’s one of the easiest laptop upgrade methods one can consider. Thanks for revealing your ideas.

    https://businesspeopleclub.com/user/beetdegree66

  19. Thank you for sharing superb informations. Your web-site is very cool. I’m impressed by the details that you have on this blog. It reveals how nicely you perceive this subject. Bookmarked this web page, will come back for extra articles. You, my friend, ROCK! I found just the information I already searched all over the place and just could not come across. What a great web site.

    https://writeablog.net/platebridge49/discovering-the-service-collection-what-strawberry-hill-gas-engineers

  20. Fantastic goods from you, man. I’ve understand your stuff previous to and you’re just extremely excellent. I actually like what you’ve acquired here, certainly like what you’re saying and the way in which you say it. You make it enjoyable and you still care for to keep it smart. I can not wait to read far more from you. This is actually a tremendous site.

    https://youtu.be/OKZ1oi8-bCQ

  21. Great – I should certainly pronounce, impressed with your website. I had no trouble navigating through all the tabs as well as related info ended up being truly simple to do to access. I recently found what I hoped for before you know it at all. Quite unusual. Is likely to appreciate it for those who add forums or anything, site theme . a tones way for your client to communicate. Nice task..

    https://youtu.be/ZCNbm81_6rI

  22. I carry on listening to the newscast talk about receiving free online grant applications so I have been looking around for the most excellent site to get one. Could you advise me please, where could i find some?

    https://notabug.org/nutwatch8

  23. I have been browsing on-line greater than three hours lately, yet I by no means found any attention-grabbing article like yours. It is beautiful worth enough for me. Personally, if all website owners and bloggers made just right content as you probably did, the web will be much more helpful than ever before.

    http://forum.ressourcerie.fr/index.php?qa=user&qa_1=susanair8

  24. Whoa! This blog looks just like my old one! It’s on a totally different topic but it has pretty much the same page layout and design. Wonderful choice of colors!

    https://0.gp/aJhHk

  25. https://www.faceofmalawi.com/2022/06/03/nyasa-big-bullets-village-construction-set-to-commence/说道:

    You’re so awesome! I don’t suppose I’ve truly read through anything like this before. So wonderful to discover someone with a few original thoughts on this issue. Really.. thanks for starting this up. This site is one thing that is needed on the internet, someone with a little originality.

    https://hipaonline.com

  26. I truly love your website.. Excellent colors & theme. Did you develop this amazing site yourself? Please reply back as I’m attempting to create my very own website and would like to learn where you got this from or exactly what the theme is called. Kudos!

    https://lumieremusic.net

  27. When I originally commented I clicked the -Notify me when new comments are added- checkbox and already each time a comment is added I am four emails concentrating on the same comment. Will there be in whatever way you may eliminate me from that service? Thanks!

    https://portal.myskeet.com/forums/users/wastebarge12/

  28. I have to show my respect for your kindness in support of persons who should have help with this situation. Your very own commitment to getting the message around became rather advantageous and have regularly made men and women just like me to achieve their objectives. Your own warm and friendly key points means a great deal to me and extremely more to my office colleagues. Best wishes; from each one of us.

    https://unsplash.com/@traintaurus08

  29. Many individuals can be extremely active, at the same time they approach their golden years. Boating, skiing, and other activities do place you at the upper chances for being injured, but every day life is not intended as approached within an overly-cautious manner. Should you be considering a trip that features sports or activities which might be considered risky, many insurance agencies won’t cover you in case you’re injured while doing these kind of activities. Be prepared for your vacation by knowing what activities you will end up engaging in, and you may be covered however should happen.

    https://carverinnovationcenter.com/index.php?option=com_k2&view=itemlist&task=user&id=455601

  30. This would be the correct blog for anybody who would like to be made aware of this topic. You realize so much its nearly tricky to argue along with you (not too I just would want…HaHa). You certainly put a new spin using a topic thats been written about for several years. Fantastic stuff, just excellent!

    https://doodleordie.com/profile/statebridge24

  31. Hello! Do you know if they make any plugins to assist with Search Engine Optimization? I’m trying to
    get my blog to rank for some targeted keywords
    but I’m not seeing very good gains. If you know of any please
    share. Appreciate it! I saw similar art here: Escape rooms list

  32. Youre so cool! I dont suppose Ive read anything such as this just before. So nice to discover somebody with some original ideas on this subject. realy thanks for starting this up. this fabulous website is a thing that is needed on the net, someone after a little originality. beneficial problem for bringing a new challenge on the internet!

    https://www.diggerslist.com/65f66e734c84e/about

  33. Hiya very nice website!! Guy .. Excellent .. Amazing .. I will bookmark your web site and take the feeds additionally¡KI am glad to search out a lot of useful info here in the publish, we want develop extra techniques in this regard, thank you for sharing. . . . . .

    https://anyflip.com/homepage/xihvc

  34. Pingback: ananın amı
  35. This is a truly good site post. Not too many people would actually, the way you just did. I am really impressed that there is so much information about this subject that have been uncovered and you’ve done your best, with so much class. If wanted to know more about green smoke reviews, than by all means come in and check our stuff. [Reply]

    https://www.infocallp.edu.bo/profile/clefskiing31/

  36. I intended to compose you one little note to help say thanks over again on the superb tactics you’ve provided in this case. It is quite shockingly open-handed with you to deliver freely all that numerous people could have marketed for an electronic book to help make some profit on their own, chiefly seeing that you could have done it if you decided. Those strategies additionally worked like the great way to understand that someone else have the identical fervor really like my own to know lots more with respect to this condition. I know there are lots of more fun times ahead for many who see your blog post.

    https://www.webwiki.nl/brixtonhvac.co.uk

  37. Pingback: animal porn
  38. The most talked about weight loss product is finally here! FitSpresso is a powerful supplement that supports healthy weight loss the natural way. Clinically studied ingredients work synergistically to support healthy fat burning, increase metabolism and maintain long lasting weight loss. https://fitspresso-try.com/

  39. Howdy! Do you know if they make any plugins to help with Search
    Engine Optimization? I’m trying to get my website to rank for some
    targeted keywords but I’m not seeing very good results.

    If you know of any please share. Appreciate it! I saw similar article here: Link Building

  40. Hi! Do you know if they make any plugins to assist with SEO?
    I’m trying to get my blog to rank for some targeted keywords but I’m not seeing very
    good gains. If you know of any please share. Appreciate it!

    You can read similar article here: Ecommerce

  41. Very great post. I simply stumbled upon your
    blog and wanted to mention that I’ve really enjoyed surfing around your weblog
    posts. In any case I’ll be subscribing to your rss feed and I hope you write
    once more soon! I saw similar here: Ecommerce

  42. Piece of writing writing is also a fun, if you be acquainted with afterward you can write if not
    it is difficult to write. I saw similar here: Sklep online

  43. Nice blog here! Also your web site loads up fast! What host are you using?
    Can I get your affiliate link to your host? I wish my web site loaded
    up as fast as yours lol

    Feel free to visit my page; ecommerce

  44. “通常一个50 μL的反应体系内应该含有超过125g的引物”这句话中的125g 是125克吗,是不是单位错了呀

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