Sep 30, 2024
Genomic DNA Extraction from Sorted Cells
- Raining Wang1,
- Melinda Wheelock1
- 1University of Washington, Department of Genome Sciences
Protocol Citation: Raining Wang, Melinda Wheelock 2024. Genomic DNA Extraction from Sorted Cells. protocols.io https://dx.doi.org/10.17504/protocols.io.n2bvjn88wgk5/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: May 09, 2024
Last Modified: September 30, 2024
Protocol Integer ID: 99787
Funders Acknowledgement:
NHGRI (IGVF)
Grant ID: HG011969
Abstract
This protocol details the extraction of genomic DNA from cells.
Guidelines
The DNeasy columns cannot take more than 5 x 106 cells per column. Larger pellets can be split into multiple extraction reactions, then combined after elution.
Materials
Materials
● Pelleted cells for DNA extraction (thawed, if previously frozen)
● DNeasy Blood & Tissue KitsQiagenCatalog #69506
● 1.7-mL microcentrifuge tubes
● DPBS
● Monarch RNase A – 1 ml (2x0.5ml)New England BiolabsCatalog #T3018L
● Molecular grade water
● 2.0-mL microcentrifuge tubes
● 96-100% Ethanol
Equipment
● Benchtop centrifuge
● Thermomixer
● Vortexer
● Picofuge
● Nanodrop
Extraction of genomic DNA from cells
Extraction of genomic DNA from cells
39m
39m
Prepare a lysis master mix of the following reagents and mix by vortexing briefly:
| A | B | C | |
| Reagent | Volume per sample (uL) | Volume for X samples (uL) | |
| DPBS | 100 | X * 100 * 1.05 | |
| Buffer AL | 200 | X * 200 * 1.05 | |
| Proteinase K | 20 | X * 20 * 1.05 | |
| RNase A | 2 | X * 2 * 1.05 | |
| Total | 322 |
Aliquot 322 µL of this master mix into labeled 1.7-mL microcentrifuge tubes.
Resuspend the pellets in 100 µL DPBS for every 5 x 106 cells in each sample pellet.
- If you have 10 x 106 cells in your pellet, resuspend in 200 µL , then split into 2 separate tubes.
Add resuspended cells in 100 µL DPBS to an appropriately labeled tube containing the lysis master mix. Mix by vortexing.
Incubate the resuspended cells at 56 °C with agitation at 1500 rpm, 00:30:00 .
30m
Spin down the lysis tubes briefly, then add 200 µL of 96-100% ethanol to the lysed cells. Mix thoroughly by vortexing. Spin the tubes down to remove drops from lids.
Transfer the lysed cell mixture to a labeled DNeasy mini spin column in a 2mL collection tube (both provided in the kit).
Centrifuge at 6000 x g, 00:01:00 . Discard the flow-through and collection tube.
1m
Place the spin column in a new 2mL collection tube and add 500 µL Buffer AW1 (provided in kit) to the column. Centrifuge at 6000 x g, 00:01:00 . Discard the flow-through and collection tube.
1m
Place the spin column in a new 2 mL collection tube and add 500 µL Buffer AW2 (provided in kit) to the column. Centrifuge at 20000 x g, 00:03:00 . Discard the flow-through and collection tube.
3m
Place the spin column in a new 2mL collection tube and centrifuge at 20000 x g, 00:01:00 .
1m
Place the spin column in a new (labeled) 1.7-mL microcentrifuge tube.
Add 100 µL of molecular grade water to the spin column and incubate at Room temperature for 00:02:00 . Centrifuge at 6000 x g, 00:01:00 .
3m
Repeat Step 13 with an additional 100 µL of molecular grade water.
Transfer any split samples (pellets with more than 5 x 106 cells) into labeled 2.0mL microcentrifuge tubes.
Quantitate with the Nanodrop, and store the samples at -20 °C .