Sep 23, 2022
Genomic DNA isolation from fixed cells
- 1Department of Biochemistry, Stanford University
Protocol Citation: Herschel Dhekne, Suzanne R Pfeffer, Ebsy Jaimon 2022. Genomic DNA isolation from fixed cells. protocols.io https://dx.doi.org/10.17504/protocols.io.eq2lynm9qvx9/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: August 18, 2022
Last Modified: May 31, 2024
Protocol Integer ID: 68847
Keywords: DNA isolation, Proteinase K, Econospin, ASAPCRN
Funders Acknowledgements:
Aligning Science Across Parkinson's Disease
Grant ID: ASAP-000463
Abstract
This protocol details the procedure of genomic DNA isolation from fixed cells.
Attachments
iugzbc32f.docx
14KB
Materials
Reagents:
- Proteinase K (Zymo #D3001-2-20) (20mg/ml stock in storage buffer).
- RNase A (Sigma #70856) (10mg/ml stock).
- 100% Molecular biology grade Ethanol.
- Silica spin columns (Econospin).
- Qiagen buffer AL.
- Qiagen buffer AW1.
- Qiagen buffer AW2.
- Nuclease free water.
Buffer AL (storage: room temperature (RT))
| A | B | |
| Tris-HCl (7.4) | 50 mM | |
| Guanidine HCl | 5.5 M | |
| EDTA | 20 mM | |
| Triton X-100 | 1.3% |
Buffer AW1 (storage: RT)
| A | B | |
| Guanidine HCl | 1 M | |
| EtOH, pH 5.5 | 57% |
Buffer AW2 / PE (storage: RT)
| A | B | |
| Tris-HCl (pH 7.5) | 10 mM | |
| Ethanol | 80% |
Buffer AW2 / PE (storage: RT)
| A | B | |
| Tris-HCl (pH 9) | 10 mM | |
| EDTA | 0.5 mM |
Genomic DNA isolation from fixed cells
Genomic DNA isolation from fixed cells
38m 5s
38m 5s
Resuspend 1-3 X106 cells in 250 µL of PBS and transfer to a 2 mL tube (this would represent one 3.5 cm dish of 3T3 cells).
Add 200 µg Proteinase K (from a 20 mg/mL stock) and 200 µg RNase A (from a 20 mg/mL stock).
Incubate cells at 37 °C for 00:30:00 in a water bath.
30m
Add 250 µL Qiagen AL lysis buffer per 250 µL of the protease and RNAase-containing cell suspension and mix thoroughly.
Place tubes in an incubator at 56 °C with shaking at 800 rpm Overnight , capped.
Note
Each tube is parafilm sealed to ensure safety.
Add 250 µL , 100% molecular biology grade ethanol; mix slowly using a slow vortex for 00:00:05 .
5s
With a razor blade, trim the tip of a 1 mL pipet tip to enlarge the opening. Use this tip to pipet out DNA from the ethanol solution and apply it onto a silica spin DNA binding column (e.g. EconoSpin 1920-250).
Spin at 6000 x g for 00:01:00 in a fixed angle tabletop microfuge; aspirate and discard flow-through.
1m
Add 500 µL Qiagen buffer AW1 to the column, spin again at 6000 x g for 00:01:00 , aspirate and discard flow-through.
1m
Add 500 µL Qiagen buffer AW2, spin at 8000 x g for 00:01:00 , aspirate and discard flow-through.
1m
Spin once more using microfuge to remove excess ethanol at 13000 x g for 00:01:00 .
1m
Transfer column into a new, 1.5 mL collection tube.
Elute with pre-warmed, 100 µL nuclease-free water or TE.
Note
Volume depends on starting number of cells: use 100 µL per 1 million cells.
Incubate for 00:01:00 , then spin at 13000 x g for 00:01:00 .
2m
Add another 50 µL nuclease free water to accomplish a second elution.
Incubate at Room temperature for 00:01:00 , then spin as before at 13000 x g for 00:01:00 .
2m
The two flow-through fractions contain the genomic DNA.
Perform Nanodrop and Qubit HS DNA estimation to calculate yield.
Note
Theoretically, 1X106cells should yield 6 µg DNA.