欢迎您, 登录 | 注册

首页 | English

X
加载中

This is a simple protocol for isolating genomic DNA from fresh plant tissues. DNA from this experiment can be used for all kinds of genetics studies, including genotyping and mapping. This protocol uses Edward’s extraction buffer to isolate DNA.

Thanks for your further question/comment. It has been sent to the author(s) of this protocol. You will receive a notification once your question/comment is addressed again by the author(s).
Meanwhile, it would be great if you could help us to spread the word about Bio-protocol.

X

[Bio101] Extract Genomic DNA from Arabidopsis Leaves (Can be Used for Other Tissues as Well)
[Bio101] 拟南芥叶片基因组DNA的提取 (其它组织也适用)

分子生物学 > DNA > DNA 提取
作者: Yongxian Lu
Yongxian LuAffiliation: Carnegie Institution for Science, Stanford University, Stanford, USA
For correspondence: yxlu@stanford.edu
Bio-protocol author page: a28
7/5/2011, 14780 views, 1 Q&A
DOI: https://doi.org/10.21769/BioProtoc.90

[Abstract] This is a simple protocol for isolating genomic DNA from fresh plant tissues. DNA from this experiment can be used for all kinds of genetics studies, including genotyping and mapping. This protocol uses Edward’s extraction buffer to isolate DNA.

[Abstract]

Materials and Reagents

  1. Ethanol (EtOH)
  2. Isopropanol
  3. NaCl
  4. EDTA
  5. SDS
  6. Tris (pH 7.5)
  7. Edward buffer (see Recipes)

Equipment

  1. Ceramic mortar and pestles
  2. Centrifuges (Eppendorf)
  3. Vortex (VWR International)

Procedure

  1. Add 400 μl Edward extraction buffer to a 1.5 μl tube.
  2. Take 2-3 small leaves or 1 big leaf, grind with pestle (2-3 weeks old).
  3. Vortex 5 sec, set at room temperature until all preps are ready.
  4. Spin at 16,000 rpm for 2 min.
  5. Transfer 300 μl of suspension to a fresh tube.
  6. Add 300 μl of isopropanol at room temperature for 2 min.
  7. Spin 5 min, wash pellet with 70% EtOH, and dry at room temperature.
  8. Resuspend in 100 μl H2O and store at -20 °C.
  9. Use 2 - 4 μl for PCR reaction to validate the presence of target DNA.

Recipes

  1. Edward buffer
    200 mM Tris (pH 7.5)
    250 mM NaCl
    25 mM EDTA
    0.5% SDS

References

  1. Lu, Y., Chanroj, S., Zulkifli, L., Johnson, M. A., Uozumi, N., Cheung, A. and Sze, H. (2011). Pollen tubes lacking a pair of K+ transporters fail to target ovules in Arabidopsis. Plant Cell 23(1): 81-93.

材料和试剂

 

1.       乙醇

2.       异丙醇

3.       氯化钠

4.       EDTA(乙二胺四乙酸)

5.       SDS (十二烷基磺酸钠)

6.       Tris pH7.5

 

仪器

 

1.       研钵及

2.       台式离心机 (Eppendorf)

3.       振荡器 (VWR)

 

步骤

 

1.       1.5mL的离心管里加入400mL Edward提取溶液。

2.       在加入提取液的离心管里将2-3片小叶或1片大叶用棒磨碎  [2-3周的植株]

3.       振荡器上振荡5秒,室温放置直到其它准备工作准备完毕。

4.       室温16000rpm离心2分钟。.

5.       300 mL离心后的上清液转移到干净的离心管里。

6.       加入300 mL异丙醇,混匀后室温放置2分钟。

7.       室温16000rpm离心5分钟,去上清后用70%的乙醇洗沉淀,室温下干燥沉淀

8.       100 mL去离子水溶解DNA,贮存在–20 °C冰箱里。

9.       吸取2-4 mLDNA溶液用于PCR反应。

 

配方

 

1.       Edward 提取溶液:

200 mM Tris pH7.5

250 mM NaCl (氯化钠)

25 mM EDTA (乙二胺四乙酸)

0.5% SDS (十二烷基磺酸钠)

 

参考文献

 

1.        Lu Y., Chanroj S., Zulkifli L., Johnson M.A., Uozumi N., Cheung A., Sze H. (2011). Pollen tubes lacking a pair of K+ transporters fail to target ovules in Arabidopsis. Plant Cell 23(1): 81-93. 

 

English
中文翻译

免责声明

为了向广大用户提供经翻译的内容,www.bio-protocol.org 采用人工翻译与计算机翻译结合的技术翻译了本文章。基于计算机的翻译质量再高,也不及 100% 的人工翻译的质量。为此,我们始终建议用户参考原始英文版本。 Bio-protocol., LLC对翻译版本的准确性不承担任何责任。

X


How to cite this protocol: Lu, Y. (2011). Extract Genomic DNA from Arabidopsis Leaves (Can be Used for Other Tissues as Well). Bio-protocol Bio101: e90. DOI: 10.21769/BioProtoc.90; Full Text



可重复性反馈:

  • 添加图片
  • 添加视频

我们的目标是让重复别人的实验变得更轻松,如果您已经使用过本实验方案,欢迎您做出评价。我们鼓励上传实验图片或视频与小伙伴们(同行)分享您的实验心得和经验。(评论前请登录)

问题&解答:

  • 添加图片
  • 添加视频

(提问前,请先登陆)bio-protocol作为媒介平台,会将您的问题转发给作者,并将作者的回复发送至您的邮箱(在bio-protocol注册时所用的邮箱)。为了作者与用户间沟通流畅(作者能准确理解您所遇到的问题并给与正确的建议),我们鼓励用户用图片或者视频的形式来说明遇到的问题。由于本平台用Youtube储存、播放视频,作者需要google 账户来上传视频。


登陆 | 注册
8/12/2014 6:34:03 PM  

Marisa Rosa
Gregor Mendel Institute of Molecular Plant Biology, Austria

Should it not be ul instead of ml throughout the procedures?

8/12/2014 11:37:05 PM  

Yongxian Lu (Author)
Carnegie Institution for Science, Stanford University, USA

Yes, you are right. It should be ul, instead of ml. Guess it's a typo by the editorial team. sorry for that.

10/10/2016 11:48:23 AM  

Jianxin Zhao
University of Florida

Can this method could be used for extraction of Medicago or red clover ?

Reply

Please login to post your questions/comments. Your questions will be directed to the authors of the protocol. The authors will be requested to answer your questions at their earliest convenience. Once your questions are answered, you will be informed using the email address that you register with bio-protocol.
You are highly recommended to post your data (images or even videos) for the troubleshooting. For uploading videos, you may need a Google account because Bio-protocol uses YouTube to host videos.

Login | Register

引用格式
分享
Twitter Twitter
LinkedIn LinkedIn
Google+ Google+
Facebook Facebook