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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.

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Micrococcal Nuclease (MNase) Assay of Arabidopsis thaliana Nuclei

Cell Biology > Organelle isolation > Nuclei
Authors: Laia Armengot
Laia ArmengotAffiliation: Departament de Bioquímica i Biologia Molecular, Universitat Autònoma de Barcelona, Bellaterra, Barcelona, Spain
For correspondence: laia.armengot@uab.cat
Bio-protocol author page: a530
 and Jordi Moreno-Romero
Jordi Moreno-RomeroAffiliation: Departament de Bioquímica i Biologia Molecular, Universitat Autònoma de Barcelona, Bellaterra, Barcelona, Spain
For correspondence: jordi.moreno@slu.se
Bio-protocol author page: a529
Vol 3, Iss 7, 4/5/2013, 5226 views, 2 Q&A, How to cite
DOI: https://doi.org/10.21769/BioProtoc.455

[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, Nuclei extraction, Microccocal nuclease digestion, Chromatin density

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.


How to cite: 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; Full Text



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2/1/2016 1:38:12 AM  

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 11:54:15 PM  

Jordi Moreno-Romero (Author)
Departament de Bioquímica i Biologia Molecular,Universitat Autònoma de Barcelona

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.

Reply

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7/9/2015 8:46:11 AM  

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 8:56:54 AM  

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.

7/10/2015 8:17:51 AM  

Jordi Moreno-Romero (Author)
Departament de Bioquímica i Biologia Molecular,Universitat Autònoma de Barcelona

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 12:01:16 PM  

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

7/11/2015 10:52:53 AM  

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




8/11/2015 6:42:04 AM  

Jordi Moreno-Romero (Author)
Departament de Bioquímica i Biologia Molecular,Universitat Autònoma de Barcelona

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

Reply

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