Materials and Reagents

Tris-HCl (pH 7.4)
Glycerol
KCl
EDTA (pH 7.5)
MgCl₂
Sucrose
Triton X-100
Murashige and Skoog basal medium (Sigma-Aldrich, catalog number: M0404-10L )
Phenylmethanesulfonylfluoride (PMSF)
Dithiothreitol (DTT)
Proteinase inhibitor (PI) (complete EDTA-free) (Roche Diagnostics, catalog number: 04693132001 )
Phytagel (Sigma-Aldrich, catalog number: P8169-1KG )
Liquid nitrogen
Lysis buffer (LB) (see Recipes)
Nuclei resuspension buffer with 0.2% Triton X-100 (NRBT) (see Recipes)
Nuclei resuspension buffer (NRB) (see Recipes)
Nuclei storage buffer (NSB) (see Recipes)
MS (see Recipes)

Equipment

Centrifuges (e.g. Eppendorf centrifuge 5810 R that can be refrigerated and will hold 50 ml tubes)
Mortar and pestle
100 μm and 40 μm nylon mesh (BD Biosciences, Falcon^®, catalog number: REF352360 , REF352340 )
50 ml conical tube

Procedure

Grow Arabidopsis seeds on MS for 2 weeks or on soil for 4 weeks. Collect approximately 1 g of Arabidopsis tissue (seedlings of about 50 plate-grown plants, leaves of approximately 20 soil-grown plants), freeze in liquid nitrogen, and then follow the steps listed below.
Note: Always keep the sample on ice.

Grind the tissue to a fine powder in liquid nitrogen using a cold mortar and pestle. Collect the powder into a 50 ml conical tube.
Add 2 ml cold Lysis buffer into the powder and homogenize the mixture by gentle shaking or pipetting.
Note: If the sample is frozen from excess liquid nitrogen, wait until it is thawed enough that it can be homogenized.
Filter the homogenate through a 100 μm and 40 μm nylon mesh sequentially.
Centrifuge the filtered homogenate at 1,500 x g at 4 °C for 10 min to pellet the nuclei.
Discard the supernatant and add 3 ml NRBT to the pellet. Re-suspend the nuclei by pipetting.
Centrifuge the sample at 1,500 x g at 4 °C for 10 min. Repeat step 5 and 6 twice more.
Discard the supernatant and add 3 ml NRB to the pellet. Re-suspend the nuclei by pipetting.
Centrifuge at 1,500 x g at 4 °C for 10 min to pellet the nuclei, and discard the supernatant. The purpose of this step is to remove the Triton X-100. The nuclei can now be used for any purpose. For example, they can be used to detect nuclear protein using a western blot. If the nuclei cannot used immediately, they should be re-suspended in 400 μl NSB buffer, quickly frozen in liquid N₂, and stored at -80 °C. The nuclei can last for at least half a year.

Recipes

Lysis buffer (LB)
20 mM Tris-HCl (pH 7.4) 2 ml (2 M)
25% glycerol 25 ml
20 mM KCl 1 ml (2 M)
2 mM EDTA 0.4 ml (0.5 M)
2.5 mM MgCl₂ 0.25 ml (1 M)
250 mM sucrose 12.5 ml (2 M)
Add H₂O to 100 ml
Add DTT and PMSF to a final concentration of 1 mM respectively, immediately before use.
Nuclei resuspension buffer with 0.2% Triton X-100 (NRBT)
20 mM Tris-HCl (pH 7.4) 2 ml (1 M)
25% glycerol 25 ml
2.5 mM MgCl₂ 0.25 ml (1 M)
0.2% Triton X-100 0.2 ml
Add H₂O to 100 ml
Nuclei resuspension buffer (NRB)
20 mM Tris-HCl (pH 7.4) 2 ml (1 M)
25% Glycerol 25 ml
2.5 mM MgCl₂ 0.25 ml (1 M)
Add H₂O to 100 ml
Nuclei storage buffer (NSB)
20 mM Tris-HCl (pH 7.4) 2 ml (1 M)
25% Glycerol 25 ml
2.5 mM MgCl₂ 0.25 ml (1 M)
Sucrose 15.1 g
Add H₂O to 100 ml
Add PI before use (1/100 dilution)
MS
Sucrose 10 g
Murashige and Skoog basal medium 4.4 g
Phytagel 3 g
Add H₂O to 1 L, adjust pH to 5.6-5.8 using KOH and autoclave for 30 min.

Acknowledgments

This protocol was adapted from the research article Xu et al. (2012). We are grateful for financial support from Natural Sciences and Engineering Research Council of Canada (NSERC) Discovery program and the 973 program of the Chinese Ministry of Science and Technology, grant number 2011CB10070.

References

Cheng, Y. T., Germain, H., Wiermer, M., Bi, D., Xu, F., Garcia, A. V., Wirthmueller, L., Despres, C., Parker, J. E., Zhang, Y. and Li, X. (2009). Nuclear pore complex component MOS7/Nup88 is required for innate immunity and nuclear accumulation of defense regulators in Arabidopsis. Plant Cell 21(8): 2503-2516.
Xu, F., Xu, S., Wiermer, M., Zhang, Y. and Li, X. (2012). The cyclin L homolog MOS12 and the MOS4-associated complex are required for the proper splicing of plant resistance genes. Plant J 70(6): 916-928.