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Micrococcal Nuclease (MNase) Assay of Arabidopsis thaliana Nuclei
微球菌核酸酶(Mnase)试验分析拟南芥染色质结构   

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Abstract

Micrococcal nuclease (MNase) is able to produce double-strand breaks within nucleosome linker regions. The efficiency of MNase digestion depends on the degree of chromatin compaction, being more easily digested the regions of less compacted chromatin. The MNase protocol described here can be used to asses changes in the chromatin structure of nuclei extracted from Arabidopsis seedlings.

Keywords: Arabidopsis thaliana(拟南芥), Nuclei extraction(细胞核提取), Microccocal nuclease digestion(microccocal核酸酶消化), Chromatin density(染色质密度)

Materials and Reagents

  1. Micrococcal Nuclease (MNase) (Roche Applied Science, catalog number: 10107921001 , 15,000 U)
  2. Proteinase K (Roche Applied Science, catalog number: 03115836001 )
  3. PIPES (Sigma-Aldrich, catalog number: P-9291 )
  4. Liquid nitrogen
  5. Sucrose
  6. KCl
  7. MgCl2
  8. CaCl2
  9. Triton X-100
  10. 1 mM PMSF (freshly added)
  11. Tris/HCl (pH 7.8)
  12. EDTA
  13. SDS
  14. Ethidium bromide
  15. Nuclei extraction buffer A, B, C (see Recipes)
  16. MNase buffer (see Recipes)
  17. 2x stop buffer  (see Recipes)
  18. 10x Proteinase K  buffer (see Recipes)

Equipment

  1. Pestle and mortar
  2. Centrifuges
  3. 37 °C oven
  4. 70 μm Nylon mesh
  5. 50 ml Falcon tubes
  6. Centrifuge tubes
  7. 2 ml microtubes
  8. Nylon mesh

Procedure

  1. Grind frozen Arabidopsis plantlets (2 g) with a pestle and a mortar (previously cooled with liquid nitrogen) under liquid nitrogen.
    Note: Perform the following steps on ice.
  2. Add the homogenized plant material to 10 ml of nuclei extraction buffer A in a 50 ml Falcon tube. Mix it well by vortexing.
  3. Filter twice the plant homogenate obtained in step 2, using a 70 μm nylon mesh placed in a funnel.
    Note: perform the second filtering step in the centrifuge tubes needed for step 4.
  4. Centrifuge at 10,000 x g for 20 min at 4 °C.
  5. Discard the supernatant by decantation.
    Note: Discard all the supernatant by pipetting.
  6. Resuspend the pellet in 200-500 μl of nuclei extraction buffer B (this volume can be adjusted depending on the size of the pellet).
  7. Pipet 200-500 μl of nuclei extraction buffer C (it must be the same volume as the one used in step 6) and place it into an empty 2 ml Eppendorf tube.
    Note: Nuclei extraction buffer C is viscous because of its high sucrose content. You should perform this step slowly in order to avoid the formation of bubbles.
  8. Add the resuspended pellet from step 6 onto the layer of buffer C obtained in step 7.
    Note: Pay attention in not disturbing the layer of Buffer C when you add the resuspended pellet.
  9. Centrifuge at 12,000 x g for 1 h at 4 °C.
  10. Discard all the supernatant by pipetting.
  11. Resuspend the pellet in 250 μl of MNase buffer (this volume can be adjusted depending on the size of the pellet).
    Note: At this point you can add 35% glycerol to the nuclei samples, submerge them into liquid nitrogen, and keep frozen at -80 °C until MNase digestion is going to be performed. At that moment, nuclei are defrozen and centrifuged at 16,000 x g for 20 min at 4 °C in order to remove the glycerol. Finally, the nuclei are resuspended in MNase buffer.
  12. Quantify the DNA concentration by measuring the absorbance at 260 nm. Usually a dilution 1:20 should be used.
    Optional: Analyze the DNA integrity before performing the MNase digestion. For this purpose, mix equal volumes of nuclei and 2x stop buffer, and centrifuge at 16,000 x g for 10 min at 4 °C. Analyze the supernatant on a 1.2% agarose gel stained with ethidium bromide.


    Figure 1. Analysis of DNA integrity before performing the MNase digestion. Several procedures were used for this purpose: (A) mixing equal volums of nuclei and milliQ water; (b) mixing equal volumes of nuclei and 2x stop buffer and, (B) mixing equal volumes of nuclei and 2x stop buffer, and centrifugation as described in the protocol. The procedure in (C) gave the best results and was used routinely.

  13. Perform this step if you want to compare different samples, if not, you can directly proceed to MNase digestion (step 14). Dilute the sample(s) with MNase buffer in order to obtain the same DNA concentration in all of them (usually a range of concentrations between 300 ng/μl and 600 ng/μl).
    Note: The final volume for all the samples must be the same and it must be adjusted depending on the number of MNase digestions you want to perform. As a rule, use between 20 μl and 40 μl per reaction.
    To study chromatin sensitivity to MNase, two complementary protocols can be used: Digestion with different concentrations (Units) of MNase (step 14) and/or digestion with a desired MNase concentration during different incubation times (step 15).
  14. Incubate the suspension of nuclei from step 13 with different Units of MNase, at 37 °C for 15 min. For instance, you can use 1, 2.5, 5, 10, 20, 40 U/ml or higher concentrations, until you observe total degradation of the high molecular weight DNA band (Figure 2). For this purpose, prepare the MNase solutions by making serial dilutions from the enzyme stock (for example 10,000 U/ml), in order to add the same volume of MNase to each individual reaction. For example, aliquot 30 μl of nuclei and add 10 μl of MNase at the desired concentration. To stop the reaction, add 40 μl of 2x stop buffer. Finally, add Proteinase K 1x buffer and  1 μl of Proteinase K (stock 10 mg/ml), and incubate overnight at 37 °C.
  15. Incubate the diluted nuclei with the MNase at 4 °C (*) for different periods of time, in order to perform time-course studies at a desired concentration of MNase. At each time point, transfer 20 μl of the reaction to an eppendorf tube containing 20 μl of stop buffer 2x and mix. Add Proteinase K 1x buffer and 1 μl of Proteinase K (stock 10 mg/ml), and incubate overnight at 37 °C.
    (*) The optimal digestion temperature of MNase is 37 °C. At 4 °C MNase digestion is slower than at 37 °C, so you can use longer incubation time of digestion at 4 °C than at 37 °C.
    Note: RNase treatment can be performed at the end of step 14 and step 15 if you observe RNA contamination in your samples.
  16. Add DNA loading buffer to the sample obtained in step 14 and step 15 and visualize the results by electrophoresis on 1.2% agarose gels stained with ethidium bromide.


    Figure 2. Example of MNase digestion using diferent concentrations of MNase. Same amounts of nuclei were digested with increasing concentrations of MNase at 37 ºC for 15 min and analysed in an 1.2% agarose gel.

Recipes

  1. Nuclei extraction buffer A
    0.25 M sucrose
    60 mM KCl
    15 mM MgCl2
    1 mM CaCl2
    15 mM PIPES (pH 6.8)
    0.8% Triton X-100
    1 mM PMSF (freshly added)
  2. Nuclei extraction buffer B
    0.25 M sucrose
    10 mM Tris/HCl (pH 8.0)
    10 mM MgCl2
    1% v/v Triton X-100
    5 mM β-mercaptoethanol (freshly added)
    1 mM PMSF (freshly added)
  3. Nuclei extraction buffer C
    1.7 M sucrose
    10 mM Tris/HCl (pH 8.0)
    10 mM MgCl2
    0.5% Triton X-100
    5 mM β -mercaptoethanol
    1 mM PMSF
  4. MNase buffer
    0.3 M sucrose
    20 mM Tris/HCl (pH 7.5)
    3 mM CaCl2
  5. 2x stop buffer
    50 mM EDTA
    1% SDS
  6. 10x Proteinase K buffer
    100 mM Tris/HCl (pH 7.8)
    50 mM EDTA
    5% SDS

Acknowledgments

This protocol was developed for the work previously published in Plant Journal (Moreno-Romero et al., 2012). This work was supported by grants BFU2007-60569, BFU2010-15090 and Consolider Ingenio 2010 CSD2007-00036 from the Ministerio de Educación y Ciencia, (Spain) and grants 2005SGR-00112 and 2009SGR-795 from the Generalitat de Catalunya, Catalunya (Spain). L.A. and J.M.-R. were recipients of fellowships from the Ministerio de Educación y Ciencia (Spain) and the Universitat Autònoma de Barcelona respectively.

References

  1. Moreno-Romero, J., Armengot, L., Mar Marques-Bueno, M., Britt, A. and Carmen Martinez, M. (2012). CK2-defective Arabidopsis plants exhibit enhanced double-strand break repair rates and reduced survival after exposure to ionizing radiation. Plant J 71(4): 627-638.

简介

微球菌核酸酶(MNase)能够在核小体接头区域内产生双链断裂。 MNase消化的效率取决于染色质压实的程度,更容易消化较少压实的染色质的区域。 这里描述的MNase方案可以用于评估从拟南芥幼苗提取的核的染色质结构的变化。

关键字:拟南芥, 细胞核提取, microccocal核酸酶消化, 染色质密度

材料和试剂

  1. 微球菌核酸酶(MNase)(Roche Applied Science,目录号:10107921001,15,000U)
  2. 蛋白酶K(Roche Applied Science,目录号:03115836001)
  3. PIPES(Sigma-Aldrich,目录号:P-9291)
  4. 液氮
  5. 蔗糖
  6. KCl
  7. MgCl 2
  8. CaCl <2>
  9. Triton X-100
  10. 1mM PMSF(新鲜加入)
  11. Tris/HCl(pH7.8)
  12. EDTA
  13. SDS
  14. 溴化乙锭
  15. 核提取缓冲液A,B,C(参见配方)
  16. MNase缓冲区(参见配方)
  17. 2x停止缓冲区 (见配方)
  18. 10x蛋白酶K 缓冲区(请参阅配方)

设备

  1. 杵和臼
  2. 离心机
  3. 37℃烘箱
  4. 70μm尼龙网
  5. 50ml Falcon管
  6. 离心管
  7. 2 ml微量管
  8. 尼龙网

程序

  1. 在液氮下用研杵和研钵(预先用液氮冷却)研磨冷冻的拟南芥苗(2g)。
    注意:在冰上执行以下步骤。
  2. 将均质化的植物材料加入到50ml的Falcon管中的10ml核提取缓冲液A. 通过涡旋混合好。
  3. 使用放置在漏斗中的70μm尼龙筛过滤两次步骤2中获得的植物匀浆 注意:在步骤4所需的离心管中执行第二个过滤步骤。
  4. 在4℃下以10,000×g离心20分钟
  5. 通过倾析弃去上清液。
    注意:通过移液除去所有的上清液。
  6. 将沉淀重悬在200-500μl的核提取缓冲液B中(该体积可以根据沉淀的大小进行调整)。
  7. 吸取200-500μl的核提取缓冲液C(它必须与步骤6中使用的体积相同),并将其放入空的2ml Eppendorf管中。
    注意:核提取缓冲液C是粘稠的,因为其高蔗糖含量。您应该慢慢执行此步骤,以避免形成气泡。
  8. 将来自步骤6的重悬沉淀物加入到步骤7中获得的缓冲液C层上 注意:在添加重新悬浮的颗粒时,请注意不要打扰缓冲液C的层。
  9. 在4℃下,12,000小时 g离心1小时。
  10. 通过移液除去所有上清液。
  11. 将沉淀重悬于250μlMNase缓冲液中(此体积可根据沉淀的大小进行调整)。
    注意:在这一点上,你可以添加35%甘油到核的样品,将它们浸没在液氮中,并保持在-80°C冷冻,直到MNEs消化将要进行。此时,将细胞核解冻并在4℃下以16,000×g离心20分钟以除去甘油。最后,将核重悬在MNase缓冲液中。
  12. 通过测量260 nm处的吸光度来定量DNA浓度。通常应使用1:20的稀释倍数 可选:在进行MNase消化之前分析DNA完整性。为此目的,混合等体积的核和2×终止缓冲液,并在4℃下以16,000×g离心10分钟。在用溴化乙锭染色的1.2%琼脂糖凝胶上分析上清液。


    图1.在进行MNase消化之前分析DNA完整性为此目的使用几种方法:(A)混合等体积的核和milliQ水; (b)混合等体积的核和2×终止缓冲液,和(B)混合等体积的核和2×终止缓冲液,并如方案中所述进行离心。 (C)中的程序给出了最好的结果,并且常规使用
  13. 如果要比较不同的样品,执行此步骤,如果不是,可以直接进行MNase消化(步骤14)。用MNase缓冲液稀释样品,以获得所有样品中相同的DNA浓度(通常浓度范围在300 ng /μl和600 ng /μl之间)。
    注意:所有样品的最终体积必须相同,必须根据要执行的MNase消化数量进行调整。通常,每次反应使用20μl至40μl。
    为了研究对MNase的染色质敏感性,可以使用两种补充方案:在不同孵育时间(步骤15)中,用不同浓度(单位)的MNase消化(步骤14)和/或用期望的MNase浓度消化。
  14. 孵育来自步骤13的核的悬浮液与不同单位的MNase,在37℃下15分钟。例如,您可以使用1,2.5,5,10,20,40 U/ml或更高的浓度,直到你观察到高分子量DNA条带的总降解(图2)。为此,通过从酶原液(例如10,000U/ml)制备系列稀释液来制备MNase溶液,以向每个单独的反应中加入相同体积的MNase。 例如,等分30μl的细胞核并加入10μl所需浓度的MNase。要停止反应,加入40μl的2x终止缓冲液。最后,加入蛋白酶K 1x缓冲液, 1μl蛋白酶K(原液10mg/ml),并在37℃下孵育过夜
  15. 在4℃(*)下,将稀释的核与MNase孵育不同的时间段,以便在所需浓度的MNase下进行时间 - 过程研究。在每个时间点,将20μl的反应转移到含有20μl终止缓冲液2x的eppendorf管中并混合。加入蛋白酶K 1×缓冲液和1μl蛋白酶K(原液10mg/ml),并在37℃下孵育过夜。
    (*)MNase的最适消化温度为37℃。在4°C MNase消化比在37°C慢,所以你可以使用更长的消化的孵育时间在4°C比在37°C。
    注意:如果观察到样品中的RNA污染,可以在步骤14和步骤15结束时进行RNA酶处理。
  16. 向步骤14和步骤15中获得的样品中加入DNA上样缓冲液并通过在用溴化乙锭染色的1.2%琼脂糖凝胶上电泳显现结果。


    图2.使用不同浓度的MNase进行MNase消化的实施例。 相同量的细胞核在浓度渐增的MNase在37℃消化15分钟,并在1.2%琼脂糖凝胶中分析。

食谱

  1. 核提取缓冲液A
    0.25 M蔗糖 60 mM KCl
    15mM MgCl 2·h/v 1mM CaCl 2
    15mM PIPES(pH 6.8)
    0.8%Triton X-100 1mM PMSF(新鲜加入)
  2. 核提取缓冲液B
    0.25 M蔗糖 10mM Tris/HCl(pH8.0)
    10mM MgCl 2/
    1%v/v Triton X-100 5mMβ-巯基乙醇(新鲜加入)
    1mM PMSF(新鲜加入)
  3. 核提取缓冲液C
    1.7 M蔗糖 10mM Tris/HCl(pH8.0)
    10mM MgCl 2/
    0.5%Triton X-100 5mMβ-巯基乙醇 1mM PMSF
  4. MNase缓冲区
    0.3 M蔗糖 20mM Tris/HCl(pH7.5) 3mM CaCl 2
  5. 2x停止缓冲区
    50mM EDTA
    1%SDS
  6. 10x蛋白酶K缓冲液
    100mM Tris/HCl(pH7.8)
    50mM EDTA
    5%SDS

致谢

该协议是为以前在Plant Journal(Moreno-Romero等人,2012年)上发表的工作开发的。 这项工作得到了西班牙教育部颁发的赠款BFU2007-60569,BFU2010-15090和Consolider Ingenio 2010 CSD2007-00036的支持,并授权来自加泰罗尼亚的Generalitat de Catalunya,Catalunya(西班牙)的2005SGR-00112和2009SGR-795。 。 L.A.和J.M.-R. 分别获得教育部长西班牙和巴塞罗那自治大学奖学金。

参考文献

  1. Moreno-Romero,J.,Armengot,L.,Mar Marques-Bueno,M.,Britt,A.and Carmen Martinez,M.(2012)。 CK2缺陷型拟南芥植物表现出增强的双链断裂修复率, 降低了暴露于电离辐射后的存活率。植物J 71(4):627-638。
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Copyright: © 2013 The Authors; exclusive licensee Bio-protocol LLC.
引用:Armengot, L. and Moreno-Romero, J. (2013). Micrococcal Nuclease (MNase) Assay of Arabidopsis thaliana Nuclei. Bio-protocol 3(7): e455. DOI: 10.21769/BioProtoc.455.
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Hyub-Bi Kim
Korea Atomic Energy Research Institute
In my experiment, I found some different result.
In gel electrophoresis, there is thick band in lower part in agarose gel (Fig 1).
In protocol, there is just a DNA band in upper part...
And also, that lower band is not disappear after MNase treatment (FIg 2).
I could see upper band of 40U was cleaved more than other when I load gel longer (50V, 1hour)as lower band was felt our and disappear (Fig 3).
I have no idea about lower thick band...
Is it normal? or something wrong in my experiment?
Please give me your opinion.

Thank you.
2/1/2016 1:38:12 AM Reply
Jordi Moreno-Romero
Departament de Bioquímica i Biologia Molecular, Universitat Autònoma de Barcelona, Spain

Dear Hyub-Bi,

In our hands we never observe this strong low molecular band you are observing in your samples. I am afraid it might be RNA contamination. Maybe, you could do a RNAse treatment at the end of steps 14 or 15.

2/1/2016 11:54:15 PM


Hyong Woo Choi
Boyce Thompson Institute for Plant Research
I tried to dissolve sucrose to make 3.7 M, but I could not fully dissolve it.
Microwaving (high temperature) make it fully dissolved, but it is crystalized as soon as cooled down...

Nuclei extraction buffer C
3.7 M sucrose
10 mM Tris/HCl (pH 8.0)
10 mM MgCl2
0.5% Triton X-100
5 mM β -mercaptoethanol
1 mM PMSF
7/9/2015 8:46:11 AM Reply
Hyong Woo Choi
Boyce Thompson Institute for Plant Research

I found that the sucrose concentration in Buffer C was actually 1.7 M, but not 3.7 M, from the original plant journal article. Please correct this typo error.

7/9/2015 8:56:54 AM


Jordi Moreno-Romero
Departament de Bioquímica i Biologia Molecular, Universitat Autònoma de Barcelona, Spain

You are right, sorry for the inconvenience caused due to the mistake. The buffer was prepared 1.7M sucrose diluting 1/2 from a 3.14M sucrose stock prepared by heating meanwhile stirring.

7/10/2015 8:17:51 AM


Hyong Woo Choi
Boyce Thompson Institute for Plant Research

Hi Jordi~! Thank you very much for your confirmation. I just finished pilot experiment to see how this protocol works.

From 2 grams of Arabidopsis leaf sample, I was able to get a big white- or grey-colored pellets, which seems like a nuclei, after the sucrose gradient centrifugation (Step 9). Nuclear pellet looks to me more than 50 uL in volume.

Do you usually can get such a large amount of nuclei pellet from 2 g Arabidopsis leaves?

And, more importantly, I was not able to measure the DNA concentration with the nanodrop at OD260 (I used 1/20 diluted samples in distilled water). It seems like to be due to the interference of white colored floating matters... (lipids???)

Could you provide a more detailed procedure how to measure DNA concentrations from different samples?

I greatly appreciated and looking forward to hearing back from you.

Sincerely,


Hyong Woo Choi

Post-Doc
Boyce Thompson Institute
533 Tower Road
Ithaca, NY 14850
e-mail:hc746@cornell.edu

7/10/2015 12:01:16 PM


Bio-protocol Editorial Team
bio-protocol.org

Hi Hyong Woo, Thank you for pointing out. The concentration of sucrose in Nuclei extraction buffer C was changed from 3.7 M to 1.7 M as requested by Dr. Jordi Moreno-Romero.

Good Luck,
Bio-protocol Editorial team




7/11/2015 10:52:53 AM


Jordi Moreno-Romero
Departament de Bioquímica i Biologia Molecular, Universitat Autònoma de Barcelona, Spain

Hi Hyong Woo,

sorry I just realized you asked for some extra information in your last request!

About the pellet size: the 50 uL gray pellet is normal with such amount of starting material. Starting with large amounts give you the possibility of try more conditions (temperatures, times, MNase concentration) but gives you a less pure pellet. Lately I am performing nuclei purification with less starting material and I am getting cleaner extractions. I also try to do a mild grinding that although decreases the efficiency but increases the purity and the integrity of the nuclei extracted.

About the NanoDrop mesurements: have you tried diluting the sample >1/20? Higher dilution should decrease the interference of cell debris and other substances. One alternative to the NanoDrop is to run an agarose gel and quantify the bands before the MNase treatment.

Hope this late answer still helps,
Sincerely, Jordi

8/11/2015 6:42:04 AM