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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, 9421 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.

方法和试剂

 

1.       cTAB (十六烷基三甲基溴化铵) (Fisher)

2.       Sodium Bisulfite 亚硫酸氢钠

3.       Sodium Chloride 氯化钠

4.       EDTA 乙二胺四乙酸

5.       Tris-HCl (pH 8.0)

6.       BME (β-巯基乙醇)

7.       氯仿

8.       异戊醇

9.       异丙醇

10.   乙醇

11.   醋酸钠

12.   醋酸

13.   TE (pH 8.0)

 

仪器

 

1.       mixer mill研磨器

2.       水浴锅

3.       离心机(96孔板转头)(Eppendorf)

 

Procedure

 

1.       组织破碎:

本步实验之前,确保植物组织是干的(空气中自然晾干或者通过抽真空处理干燥植物组织)

1)            将碳钨合金珠子加入到低温冻干的样品里,用于磨碎样品。  

2)            研磨器是通过振动连接到研磨器上的振臂,使连接到振臂上的托盘往返振动,来磨碎干叶片组织的,使用研磨器对样品进行四次研磨破碎。确保将样品研磨彻底(研磨的程度将决定DNA的产量)

3)            加入 350 ml cTAB溶液 (不要忘记加入β-巯基乙醇)。再次使用研磨器研磨样品。

4)            将破碎好的样品密封好后进行60°C 水浴处理30分钟,每隔10分钟轻柔的混匀样品。(混样前确保管盖是完全密闭的,水浴加热会使管内压升高,否则会导致样品从管内喷出)。

5)            将水浴加热后的样品放到实验台上10分钟左右,使之恢复到室温。

6)            使用台式离心机对样品进行离心处理,使粘到盖上的叶片组织离心到溶液里。

2.       抽提:

1)            向管里加入 350 ml 氯仿:异戊醇 241)(使用1的玻璃烧杯盛放氯仿:异戊醇) 在通风橱里,持续振荡离心管5分钟进行抽提。

在本步使用新的盖子。

2)            使用台式离心机在3250 rpm的转速下对样品离心20分钟。

3)            在新的管里加入200 ml 氯仿:异戊醇 241),并对新管进行相应标记。

4)            将离心后管里的上层液相(水相)加入到步骤3)的管里,大约可以得到200ml 的上层液相。

5)            在通风橱里,持续振荡离心管5分钟进行抽提。

6)            使用台式离心机在3250 rpm的转速下对样品离心20分钟。离心后的上层液相将用于后续的试验步骤里。

3.       DNA 沉淀:

1)            96孔板里加入150 ml -20°C 预冷的异丙醇。(深孔, V底)

2)            将上面步骤6)的上层液相收集后加入到含有异丙醇的对应管里。大约可以获得120-150ml 上层水相,通过轻柔翻转96孔板使溶液混匀。

3)            为了增加产量,可以使DNA-20°C 过夜沉淀。

4)            预冷台式离心机到4°C。当台式离心机的温度预冷到4°C时,使用台式离心机在3250 rpm的转速下对DNA样品离心15分钟。

5)            倒掉含有异丙醇的上清后,轻柔的将板倒置到干净的吸水纸上面。

4.       DNA漂洗:

1)            加入 500 ml 76%乙醇/0.2 M 醋酸钠溶液。  使沉淀在溶液中浸泡20分钟。.

2)            使用台式离心机在3250 rpm的转速下对样品离心10分钟。

3)            倒掉 76% 乙醇/0.2 M 醋酸钠溶液并且轻柔的将板倒置到干净的吸水纸上面。

4)            加入 250 ml 76% 乙醇/10 mM 醋酸溶液。使沉淀在溶液中浸泡2分钟。

5)            使用台式离心机在3250 rpm的转速下对样品离心10分钟。

6)            倒掉 76% 乙醇/10 mM 醋酸溶液并且轻柔的将板倒置到干净的吸水纸上面.

7)            将板正面放置到实验台上自然晾干10-15分钟或者直到晾干为止。

8)            向管内加入适量 TE (pH 8.0)以溶解DNA  可以将DNA放置到4°C过夜溶解。

a)      玉米用50ml TE溶解 (40倍浓度)

b)      拟南200ml TE溶解 (工作浓度)

9)            -20°C储存,以便用于将来使用。

 

配方

 

1.       100 ml cTAB提取溶液:

2 g cTAB (十六烷基三甲基溴化铵)

1 g 亚硫酸氢钠

28 ml 5M 氯化钠

4 ml 0.5M乙二胺四乙酸

10 ml 1.0M Tris-HCl (pH 8.0)

1.0 ml BME (β-巯基乙醇) 现用现加。

 

参考文献

 

1.        Saghai-Maroof M.A., Soliman K.M., Jorgensen R.A., Allard R.W. (1984). Ribosomal DNA spacer-length polymorphisms in barley: mendelian inheritance, chromosomal location, and population dynamics. Proceedings of the National Academy of Sciences of the United States of America 81(24): 8014-8. 

 

 

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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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