Human_Tissue_Nuclei_Isolation_Protocol_2021_10_18
Kimberly Paquette
Published: 2024-06-26 DOI: 10.17504/protocols.io.5qpvoboy7l4o/v1
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Abstract
Homebrew protocol to isolate nuclei from human frozen brain tissue.
Attachments
401-868.pdf
Steps
Stock Solutions
1.
10% Triton X-100 (100mL)
| A | B | C | D |
|---|---|---|---|
| Solution | Final Conc | Volume | Notes |
| Triton X-100 | 10% | 10 mL | |
| Ultrapure Water | Fill to: 100 mL |
2.
1Molarity (M) MgCl2 (100mL)
| A | B | C | D |
|---|---|---|---|
| Solution | Final Conc | Volume | Notes |
| MgCl2 (FW 203.31) | 1 M | 20.331 g | 203.31 g/1000 mL = 1 M 5.08275 g/100 mL = 1 M |
| Ultrapure Water | Fill to: 100 mL |
Working Solutions: PBSTA (3mL) without TritonX-100
3. *Use PBS without Calcium Chloride and Magnesium Chloride.
Note : Store at 4°C for up to 1 month.
Note
Note
| A | B | C | D |
|---|---|---|---|
| Solution | Final Conc | 1 RXN | 5.2 RXN |
| 10x PBS | 1x | 300 uL | 1560 uL |
| 1M MgCl2 | 3 mM | 9 uL | 46.8 uL |
| D-Sucrose (342.29) | 0.3 M | 0.3081 g | 1.602 g |
| ** RNasein (add on day) | 0.4 U/uL | 30 uL | 156 uL |
| Ultrapure Water | Fill to: 3 mL | Fill to: 5.6 mL |
Add most of the Ultrapure water, then PBS, then MgCl2, then Sucrose.
4.
Vortex to dissolve.
5.
Then add Ultrapure water to final volume.
6.
Add RNasein before using.
Working Solutions: 1.4 M PBS Cushion (8 mL)
7. Note : Make fresh weekly.
Note
| A | B | C | D |
|---|---|---|---|
| Solution | Final Conc | 1 RXN | 5.2 RXN |
| 10x PBS | 1x | 0.8 mL | 4.16 mL |
| 1 M MgCl2 | 3 mM | 24 uL | 124.8 uL |
| D-Sucrose | 1.4 M | 3.8336 g | 19.935 g |
| 10% Triton X-100 | 0.10% | 80 uL | 416 uL |
| Ultrapure Water | Fill to: 8 mL | Fill to: 41.6 mL |
Add half of the Ultrapure water, then PBS, then MgCl2, then Sucrose.
8.
Vortex to dissolve.
9.
Then add 10% Triton.
10.
Then add Ultrapure water to final volume.
Working Solutions: Nuclei Wash and Resuspension Buffer
11. Note: Make fresh daily.
Note
| A | B | C | D |
|---|---|---|---|
| Solution | Final Conc | 1 RXN | 5.2 RXN |
| 10x PBS | 1x | 400 uL | 2080 uL |
| 50mg/uL Ultrapure BSA | 1% | 800 uL (40 mg) | 4160 uL |
| ** RNasein (add on day) | 0.2 U/uL | 20 uL | 104 uL |
| Ultrapure Water | Fill to: 4 mL | Fill to: 20.8 mL |
Add most of the Ultrapure water, then PBS, then BSA.
12.
Tilt to dissolve, do not vortex.
13.
Add RNasein before using.
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