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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.

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[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, 15782 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(EtOH)
  2. 异丙醇
  3. NaCl
  4. EDTA
  5. >
  6. Tris(pH7.5)
  7. 爱德华缓冲区(见配方)

设备

  1. 陶瓷砂浆和杵
  2. 离心机(Eppendorf)
  3. Vortex(VWR International)

程序

  1. 加入400μl爱德华提取缓冲液到1.5μl管。
  2. 取2-3片小叶或1片大叶,用杵研磨(2-3周龄)。
  3. 涡旋5秒,设置在室温,直到所有的准备工作准备好。
  4. 以16,000rpm旋转2分钟。
  5. 转移300微升的悬浮液到一个新的管。
  6. 加入300μl异丙醇在室温下2分钟。
  7. 旋转5分钟,用70%EtOH洗涤沉淀,并在室温下干燥。
  8. 重悬在100μlH 2 O中并储存在-20℃。
  9. 使用2 - 4μl的PCR反应来验证目标DNA的存在

食谱

  1. 爱德华缓冲区
    200mM Tris(pH7.5) 250mM NaCl 25mM EDTA
    0.5%SDS

参考文献

  1. Lu,Y.,Chanroj,S.,Zulkifli,L.,Johnson,M.A.,Uozumi,N.,Cheung,A.and Sze,H。(2011)。 缺少一对K + 转运蛋白的花粉管未能靶向胚珠 拟南芥。植物细胞 23(1):81-93。
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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



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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 ?

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