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This high throughput DNA isolation protocol is used to extract DNA of high quality from plant tissues for various genetics studies, like genotyping, and mapping etc. This protocol uses the well-established CTAB extraction procedure, and has been adapted to be used with 96-well plates.

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[Bio101] DNA Extraction from Dried Plant Tissues Using 96-well format (cTab Method)
[Bio101] 高通量提取干植物组织的DNA(CTAB法-96孔板)

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, 9992 views, 5 Q&A
DOI: https://doi.org/10.21769/BioProtoc.89

[Abstract] This high throughput DNA isolation protocol is used to extract DNA of high quality from plant tissues for various genetics studies, like genotyping, and mapping etc. This protocol uses the well-established CTAB extraction procedure, and has been adapted to be used with 96-well plates.

[Abstract]

Materials and Reagents

  1. Hexadecyltrimethyl Ammonium Bromide (CTAB) (Thermo Fisher Scientific)
  2. Sodium bisulfite
  3. Tungsten carbide beads
  4. Sodium chloride
  5. EDTA
  6. Tris-HCl (pH 8.0)
  7. β-mercapto-ethanol (BME)
  8. Chloroform
  9. Octanol
  10. Isopropanol
  11. Ethanol
  12. Sodium-acetate
  13. Ammonium-acetate
  14. TE (pH 8.0)
  15. 100 ml of CTAB (see Recipes)

Equipment

  1. Centrifuges (Eppendorf)
  2. Mixer mill
  3. Glass beaker
  4. Water bath
  5. 96-square well blocks

Procedure

  1. Tissue grounding:
    Before this step, the plant tissue should have been dried (either air-dried or vacuum dried).
    1. Add tungsten carbide beads to freeze-dried samples. These will grind the sample.
    2. Grind leaf samples four times in the mixer mill, reversing orientations of the trays and switching shaker arms between grinds. Make sure the sample is ground into a fine powder (this influences yield).
    3. Add 350 μl of CTAB (don’t forget the β-mercaptoethanol). Grind the sample again in the mixer mill.
    4. Wrap the boxes with tape and incubate in the 60 °C water bath for 30 min, shaking them gently every 10 min (be sure the caps on the tubes are secure before shaking-the pressure from heating the tubes can pop them off).
    5. Sit the tubes on the bench for 10 min to allow them to return to room temperature.
    6. Spin the samples in the tabletop centrifuge for a few seconds to get the leaf tissue off the lid.

  2. Phase separation:
    1. Add 350 μl of chloroform: octanol (24:1) to the tubes (use a 1 L glass beaker for the chloroform: octanol). Shake the tubes continuously for 5 min under the fume hood.
      * Use new caps during this step.
    2. Spin the samples in the tabletop centrifuge for twenty minutes at 3,250 rpm.
    3. Add 200 μl of chloroform: octanol (24:1) to a new set of tubes and label the tubes.
    4. Remove the upper (aqueous) phase to the new tubes. Try to get about 200 μl of fluid, but less is okay.
    5. Shake the tubes continuously for 5 min under the fume hood.
    6. Spin in the tabletop centrifuge for 20 min at 3,250 rpm. The upper (aqueous) phase will be used in the following steps.

  3. DNA precipitation:
    1. Add 150 μl of -20 °C isopropanol to a set of 96-square well blocks (deep well, V-bottom).
    2. Collect the upper (aqueous) phase from tubes after step 6 in the phase separation section to the square blocks. Try to get 12-150 μl of fluid, but less is okay. Gently mix the solution by swirling the trays.
    3. To increase the yield, let the DNA precipitate overnight at -20 °C.
    4. Set the tabletop centrifuge to 4 °C. Once it has cooled down, spin the DNA samples in the tabletop centrifuge for 15 min at 3,250 rpm.
    5. Pour out isopropanol into sink and very gently tap out trays over a paper towel.

  4. DNA wash:
    1. Add 500 μl of 76% ethanol/0.2 M sodium-acetate. Let the samples sit in this solution for 20 min.
    2. Spin in the tabletop centrifuge for 10 min at 3,250 rpm.
    3. Pour out the 76% ethanol/0.2 M sodium-acetate and very gently tap out the trays over a paper towel.
    4. Add 250 μl of 76% ethanol/10 mM ammonium-acetate. Let the samples sit for 2 min.
    5. Spin in the tabletop centrifuge for 10 min at 3.250 rpm.
    6. Pour out the 76% ethanol/10 mM ammonium-acetate and very gently tap out the trays over a paper towel.
    7. Air dry on the bench for 10-15 min or until dry.
    8. Add TE (pH 8.0) to the trays. Place in the cold room at 4 °C overnight to let DNA re-suspend.
      1. 50 μl TE for corn (40x concentration)
      2. 200 μl TE for Arabidopsis (working concentration)

Recipes

  1. 100 ml of CTAB
    2 g CTAB
    1 g sodium bisulfite
    28 ml 5 M sodium chloride
    4 ml 0.5 M EDTA
    10 ml 1.0 M Tris-HCl (pH 8.0)
    1.0 ml BME right at time of use

References

  1. Saghai-Maroof, M. A., Soliman, K. M., Jorgensen, R. A. and Allard, R. W. (1984). Ribosomal DNA spacer-length polymorphisms in barley: mendelian inheritance, chromosomal location, and population dynamics. Proc Natl Acad Sci U S A 81(24): 8014-8018.

材料和试剂

  1. 十六烷基三甲基溴化铵(CTAB)(Thermo Fisher Scientific)
  2. 亚硫酸氢钠
  3. 碳化钨珠
  4. 氯化钠
  5. EDTA
  6. Tris-HCl(pH8.0)
  7. β-巯基乙醇(BME)
  8. 氯仿
  9. 辛醇
  10. 异丙醇
  11. 乙醇
  12. 醋酸钠
  13. 乙酸铵
  14. TE(pH8.0)
  15. 100ml CTAB(参见配方)

设备

  1. 离心机(Eppendorf)
  2. 混合机
  3. 玻璃烧杯
  4. 水浴
  5. 96平方井块

程序

  1. 组织接地:
    在该步骤之前,植物组织应该已经干燥(空气干燥或真空干燥)
    1. 向冷冻干燥样品中加入碳化钨珠。 这些将研磨样品。
    2. 在混合器研磨机中研磨叶样品四次,颠倒盘的方向并在研磨之间切换振动臂。 确保样品被研磨成细粉末(这会影响产量)。
    3. 加入350微升CTAB(不要忘记β-巯基乙醇)。 在混合机中再次研磨样品。
    4. 用胶带包装盒子,并在60℃水浴中孵育30分钟,每10分钟轻轻摇动(确保管子上的盖子在摇动之前是安全的 - 加热管子的压力可以弹出)。
    5. 将试管放在试验台上10分钟,使试管返回室温。
    6. 在桌面离心机中旋转样品几秒钟,以使叶组织离开盖子
  2. 相分离:
    1. 向管中加入350μl氯仿:辛醇(24:1)(使用1L玻璃烧杯用于氯仿:辛醇)。 在通风橱下连续摇动管5分钟。
      *在此步骤中使用新的上限。
    2. 在台式离心机中以3,250rpm旋转样品二十分钟。
    3. 加入200微升氯仿:辛醇(24:1)到一套新的管,并标记管。
    4. 将上层(水相)移至新管中。 尝试获得约200微升的液体,但更少是好的。
    5. 在通风橱下连续摇动管5分钟。
    6. 在台式离心机中以3,250rpm旋转20分钟。 上(水相)将用于以下步骤
  3. DNA沉淀:
    1. 加入150μl的-20°C异丙醇到一组96平方井块(深井,V-底)。
    2. 在步骤6之后,在相分离段中将来自管的上(水相)收集到方形块。 尝试获得12-150微升的流体,但更少是好的。 通过旋转轻轻混合溶液 托盘。
    3. 为了增加产量,让DNA在-20℃下沉淀过夜。
    4. 将台式离心机设置为4°C。 一旦其冷却,在台式离心机中以3,250rpm旋转DNA样品15分钟。
    5. 将异丙醇倒入水槽,轻轻地将纸盘放在纸巾上
  4. DNA洗涤:
    1. 加入500μl的76%乙醇/0.2M乙酸钠。 让样品在该溶液中静置20分钟。
    2. 在台式离心机中以3,250rpm旋转10分钟。
    3. 倒出76%乙醇/0.2M乙酸钠,轻轻地将纸盘放在纸巾上。
    4. 加入250μl的76%乙醇/10mM乙酸铵。 让样品静置2分钟。
    5. 在台式离心机中以3.250rpm旋转10分钟。
    6. 倒出76%乙醇/10mM乙酸铵,非常轻轻地将纸盘放在纸巾上。
    7. 在工作台上空气干燥10-15分钟或直到干燥。
    8. 向托盘中加入TE(pH 8.0)。 置于冷室4°C过夜,让DNA重悬。
      1. 玉米50μlTE(40x浓度)
      2. 拟南芥(工作浓度)为200μlTE

食谱

  1. 100ml CTAB
    2 g CTAB
    1克亚硫酸氢钠 28ml 5M氯化钠
    4ml 0.5M EDTA
    10ml 1.0M Tris-HCl(pH8.0) 在使用时立即使用1.0ml BME

参考文献

  1. Saghai-Maroof,M.A.,Soliman,K.M.,Jorgensen,R.A。和Allard,R.W。(1984)。 大麦中的核糖体DNA间隔长度多态性:孟德尔遗传,染色体位置和群体动力学。 Proc Natl Acad Sci USA 81(24):8014-8018。
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How to cite this protocol: Lu, Y. (2011). DNA Extraction from Dried Plant Tissues Using 96-well format (cTab Method). Bio-protocol Bio101: e89. DOI: 10.21769/BioProtoc.89; Full Text



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4/2/2015 9:48:38 PM  

ASM Islam
Tennessee State University

I want to extract dna from dry leaf samples. Will i use liquid nitrogen for grinding the dry leaf samples?in my case, I am able to grind sample finely without using liquid nitrogen. Will I keep the tubes containing grinding leaf powder in liquid nitrogen before using lysis buffer or after grinding use lysis buffer directly?

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10/24/2014 3:08:12 AM  

Ioannis Panetas
Aristotle university

Does it work with a long time dried plant?
Is it possible only with leafs or could we use οther organs of the plant?

10/31/2014 11:19:40 PM  

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

as long as the DNA is still there, it should be fine no matter how long the plants have been dried.

when use this protocol for other parts/tissues, make sure the tungsten carbide beads can crack them.

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3/29/2012 7:25:27 PM  

Please tell me. How can extract DNA from the dried leaf of a plant

4/1/2012 2:26:32 PM  

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

Even in the dry tissues, DNA is still there. So one can ground the tissue and then isolate and purify DNA.

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2/20/2012 8:30:47 PM  

Could you please explain why in the DNA wash steps (18-23) you are using acetate salts in addition to ethanol? I have checked the paper by Saghai-Maroof and they too explain nothing about it.

2/22/2012 2:44:34 AM  

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

Frankly i don't know why sodium-acetate is added to wash DNA, but my guess would be it is in order to get pure DNA by removing metabolites

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2/18/2012 5:55:45 PM  

we tried ur protocol for dried plant but we couldnt get dna band how to isolate the dan from plant

2/22/2012 2:43:54 AM  

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

This method is basically based on the classical phenol choroform DNA isolation, so it's hard to imagine that you cannot get any DNA. my suggestion would be try something like increase you sample quantity, also, pay attention to certain steps, like when you add isoproponol, make sure it mix well with the DNA solution before spinning.

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