Mar 26, 2026
Nuclei Isolation with EZ Prep Kit
- 1Yale University
Protocol Citation: Le Zhang 2026. Nuclei Isolation with EZ Prep Kit. protocols.io https://dx.doi.org/10.17504/protocols.io.36wgq7r8yvk5/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: December 09, 2021
Last Modified: March 26, 2026
Protocol Integer ID: 55790
Keywords: Nuclei Isolation, EZ Prep Kit, ASAPCRN, nuclei isolation with ez prep kit, protocol details nuclei isolation, nuclei isolation, ez prep kit, nuclei, isolation, protocol detail, protocol
Funders Acknowledgements:
ASAP
Grant ID: ASAP-000529
Abstract
This protocol details nuclei Isolation with EZ Prep Kit.
Attachments
f3xvbkx77.pdf
92KB
Materials
- EZ Prep Kit
Prepare nuclei suspension buffer by adding2 µL of RNase Inhibitor (40 U/μl ) per mL of nuclei suspension buffer. Vortex briefly to mix.
Keeping everything On ice , add 1 mL EZ PREP buffer (Sigma, NUC101) and tissue piece to the small 2 mL glass tissue grinder. Grind 3 times just to mash the tissue at the bottom of the grinder, then homogenize 25 times with pestle A. Add an additional 1 mL EZ PREP buffer and homogenize 25 times with pestle B. Move slowly to avoid spilling buffer.
Use a pipette to transfer to 15 mL conical tube. Add 2 mL EZ PREP buffer to the tissue homogenizer to rinse and pipette into conical tube. Pipette gently to mix.
Incubate On ice for 00:05:00 .
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Centrifuge for 00:05:00 at 500 x g at 4 °C .
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Remove supernatant carefully with a pipette. Do not disturb the pallet and leave behind some buffer. Resuspend in 1 mL EZ PREP buffer, then add 3 mL EZ PREP buffer to wash.
Incubate 4 °C for 00:05:00 .
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Centrifuge for 00:05:00 at 500 x g at 4 °C .
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Remove supernatant carefully with a pipette. Resuspend in 1 mL nuclei suspension buffer, then add 3 mL nuclei suspension buffer. Pipette to mix.
Note
Do not disturb the pallet and leave behind some buffer.
Centrifuge for 00:05:00 at 500 x g and 4 °C .
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Remove supernatant carefully with a pipette. Resuspend in 500 µL nuclei suspension buffer.
In a new labeled Eppendorf tube, make a 1:1 dilution of the nuclei suspension and Trypan Blue and count nuclei with hemocytometer slide (C-Chip, DHCN015).
Note
Average count x 2 (dilution in Trypan Blue) x 104 /mL.
If there are visible clumps of nuclei when counting, re-pipette gently 20 times and then incubate On ice for 00:05:00 . Make new 1:1 dilution with Trypan Blue and then recount and check for clumps of nuclei.
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