Jun 26, 2024

Public workspaceHuman_Tissue_Nuclei_Isolation_Protocol_2021_10_18

DOI
https://dx.doi.org/10.17504/protocols.io.5qpvoboy7l4o/v1
  • Kimberly Paquette1
  • 1Center for Alzheimer's and Related Dementias, National Institute on Aging, Bethesda, Maryland, USA
  • Team Lee
Jane Balster
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DOI: https://dx.doi.org/10.17504/protocols.io.5qpvoboy7l4o/v1
Protocol Citation: Kimberly Paquette 2024. Human_Tissue_Nuclei_Isolation_Protocol_2021_10_18. protocols.io https://dx.doi.org/10.17504/protocols.io.5qpvoboy7l4o/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: April 11, 2022
Last Modified: June 26, 2024
Protocol Integer ID: 60564
Keywords: nuclei from human frozen brain tissue, human frozen brain tissue, nuclei, protocol
Abstract
Homebrew protocol to isolate nuclei from human frozen brain tissue.
Attachments
Icon for 401-868.pdf
401-868.pdf
51KB
Materials
Stock Solutions

  • 10% Triton X-100 (100mL)
  • 1M MgCl2 (100mL)
Working Solutions

  • PBS
  • RNasein
  • MgCl2
  • D-Sucrose
  • Ultrapure Water
  • Ultrapure BSA



Stock Solutions
10% Triton X-100 (Amount100 mL )
ABCD
SolutionFinal ConcVolumeNotes
Triton X-10010%10 mL
Ultrapure WaterFill to: 100 mL

Concentration1 Molarity (M) MgCl2 (Amount100 mL )
ABCD
SolutionFinal ConcVolumeNotes
MgCl2 (FW 203.31)1 M20.331 g203.31 g/1000 mL = 1 M 5.08275 g/100 mL = 1 M
Ultrapure WaterFill to: 100 mL

Working Solutions: PBSTA (3mL) without TritonX-100

Note
*Use PBS without Calcium Chloride and Magnesium Chloride.

Note
Note: Store at Temperature4 °C for up to 1 month.

ABCD
SolutionFinal Conc1 RXN5.2 RXN
10x PBS1x300 uL1560 uL
1M MgCl23 mM9 uL46.8 uL
D-Sucrose (342.29)0.3 M0.3081 g1.602 g
** RNasein (add on day)0.4 U/uL30 uL156 uL
Ultrapure WaterFill to: 3 mLFill to: 5.6 mL
Add most of the Ultrapure water, then PBS, then MgCl2, then Sucrose.
Pipetting
Vortex to dissolve.
Then add Ultrapure water to final volume.
Pipetting
Add RNasein before using.
Pipetting
Working Solutions: 1.4 M PBS Cushion (8 mL)
Note
Note: Make fresh weekly.

ABCD
SolutionFinal Conc1 RXN5.2 RXN
10x PBS1x0.8 mL4.16 mL
1 M MgCl23 mM24 uL124.8 uL
D-Sucrose1.4 M3.8336 g19.935 g
10% Triton X-1000.10%80 uL416 uL
Ultrapure WaterFill to: 8 mLFill to: 41.6 mL
Add half of the Ultrapure water, then PBS, then MgCl2, then Sucrose.
Pipetting
Vortex to dissolve.
Then add 10% Triton.
Then add Ultrapure water to final volume.
Working Solutions: Nuclei Wash and Resuspension Buffer

Note
Note: Make fresh daily.
ABCD
SolutionFinal Conc1 RXN5.2 RXN
10x PBS1x400 uL2080 uL
50mg/uL Ultrapure BSA1%800 uL (40 mg)4160 uL
** RNasein (add on day)0.2 U/uL20 uL104 uL
Ultrapure WaterFill to: 4 mLFill to: 20.8 mL
Add most of the Ultrapure water, then PBS, then BSA.
Pipetting
Tilt to dissolve, do not vortex.
Add RNasein before using.
Pipetting