Mar 10, 2026
Version 2
Genomic DNA Extraction for Epigenetics Using Spin Columns V.2
- Muhammad Abbas1,2,3,
- Michael Strinden4,
- Kyle Allen4,1,2,
- Yenisel Cruz-Almeida1,2,3
- 1UF Pain Research and Intervention Center of Excellence;
- 2Department of Community Dentistry & Behavioral Science, College of Dentistry;
- 3UF Department of Neuroscience, College of Medicine;
- 4UF Department of Biomedical Engineering, College of Engineering
Protocol Citation: Muhammad Abbas, Michael Strinden, Kyle Allen, Yenisel Cruz-Almeida 2026. Genomic DNA Extraction for Epigenetics Using Spin Columns. protocols.io https://dx.doi.org/10.17504/protocols.io.dm6gp6wm1vzp/v2Version created by Michael Strinden
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: March 09, 2026
Last Modified: March 10, 2026
Protocol Integer ID: 306703
Keywords: genomic dna extraction for epigenetics, using spin columns genomic dna, spin columns genomic dna, genomic dna extraction, weight genomic dna suitable for downstream molecular application, weight genomic dna, free genomic dna, purified dna, epigenetics, mg of human knee tissue, human knee tissue, optional rnase treatment, silica spin column, rna, other methylation
Abstract
Genomic DNA was extracted from 100–300 mg of human knee tissues using bead-based mechanical homogenization in ATL lysis buffer, followed by proteinase K digestion to ensure complete tissue lysis of both soft and dense connective tissues. Lysates were processed through silica spin columns (DNeasy Blood & Tissue Kit), washed to remove contaminants, and eluted in Buffer AE, with optional RNase treatment to obtain RNA-free genomic DNA.
This protocol yields high-quality, high-molecular-weight genomic DNA suitable for downstream molecular applications. The purified DNA demonstrates appropriate concentration and purity (A260/280 and A260/230 ratios within acceptable ranges) and is ready for epigenetic sequencing workflows, including bisulfite conversion and other methylation-based assays.
Materials
DNeasy Blood & Tissue Kit (cat. no. 69504)
BeadBug‱3 Microtube homogenizer
BeadBug‱ prefilled tubes, 2.0 mL
2 ml Safe-Lock microtubes (Eppendorf, cat. no. 0030 120.094)
Pipets and pipet tips
Vortex
Centrifuge
Microcentrifuge tubes (1.5 ml or 2 ml)
Microcentrifuge with rotor for 1.5 ml and 2 ml tubes
Thermomixer, shaking water bath or rocking platform for heating at 56°C
Ethanol (96–100%)
NanoDrop and Qubit
Before start
a. All centrifugation steps are carried out at room temperature (15–25°C) in a microcentrifuge.
b. Vortexing should be performed by pulse-vortexing for 5–10 s.
c. Avoid repeated thawing and freezing of samples, as this can reduce DNA size.
d. Buffer ATL and Buffer AL may form precipitates upon storage. If necessary, warm to 56°C until the precipitates have fully dissolved.
e. Buffer AW1 and Buffer AW2 are supplied as concentrates. Before using for the first time, add the appropriate amount of ethanol (96–100%) as indicated on the bottle to obtain a working solution.
f. Preheat a thermomixer, shaking water bath or rocking platform to 56°C for use in step 2.
g. If using frozen tissue, equilibrate the sample to room temperature (15–25°C).
Setup
Pre-Extraction Processing
a. Weigh the tissue samples (100 – 300 mg) using an analytical balance.
b. Wrap the tissue samples in foil and flash-freeze them in liquid nitrogen.
c. Crush the frozen tissues using a pestle and mortar or hammer.
d. (‘Elastic’ tissues such as meniscus and cartilage can be finely minced using a scalpel before freezing to aid with homogenization).
Genomic DNA Extraction Procedure (Adapted from DNeasy Blood and Tissue Handbook)
Add ATL Lysis buffer (10 volumes per tissue mass, ~1-2 mL, e.g., 100 mg tissue in 1 mL Buffer ATL) to prefilled Bead/Bug homogenizer microtubes.
For best results, use 3 mm stainless steel beads for soft tissues (synovium, infrapatellar fatpad), and 5 mm stainless steel beads for hard tissues (medial tibial plateau, meniscus).
Transfer 100-300mg tissue to the tube.
Homogenize at maximum speed for 60 sec per cycle.
Place the tube on ice for 30 seconds between cycles to prevent overheating of both soft (synovium, infrapatellar fatpad) and hard tissues (medial tibial plateau, meniscus). Repeat for 2–3 cycles as needed. Do not exceed this time, as it may result in DNA shearing.
Centrifuge the sample briefly to ensure that all the tissue debris is on the bottom of the tube.
Add 20 µl proteinase K to the tube.
Note: Add 40 µl proteinase K if using RNAlater stabilized tissues.
Mix thoroughly by vortexing and incubate at 56°C until the tissue is completely lysed. Vortex occasionally during incubation to disperse the sample or place in a thermomixer, shaking water bath or on a rocking platform.
Lysis time varies depending on the type of tissue processed. Lysis is usually complete in 1–3 h (Soft tissues) or, for hard tissues, 6–8 h. If it is more convenient, samples can be lysed overnight; this will not affect them adversely.
Centrifuge the 2 ml microtube briefly to remove drops from inside the lid.
Optional: If RNA-free genomic DNA is required, add 4 µl RNase (100 mg/ml) and incubate for 5 min at room temperature before continuing with step 9.
Add 200 µl Buffer AL to the sample (220 µl Buffer AL if using RNAlater stabilized tissues) and mix thoroughly by vortexing. Then add 200 µl ethanol (96–100%) and mix again thoroughly by vortexing.
The sample, Buffer AL, and ethanol must be mixed immediately and thoroughly by vortexing or pipetting to yield a homogeneous solution. Buffer AL and ethanol can be premixed and added together in one step to save time when processing multiple samples. A white precipitate may form on addition of Buffer AL and ethanol. This precipitate does not interfere with the DNeasy procedure. Some samples may form a gelatinous lysate after the addition of Buffer AL and ethanol. In this case, vigorously shake or vortex the sample.
Load the mixture from step 9 (including any precipitate) into the DNeasy Mini spin column placed in a 2 ml collection tube. Centrifuge at ≥6000 x g (8000 rpm) for 1 min. Discard the flow-through and collection tube.
Place the DNeasy Mini spin column in a new 2 ml collection tube, add 500 µl Buffer AW1, and centrifuge for 1 min at ≥6000 x g (8000 rpm). Discard the flow-through and collection tube.
Place the DNeasy Mini spin column in a new 2 ml collection tube, add 500 µl Buffer AW2, and centrifuge for 3 min at 20,000 x g (14,000 rpm) to dry the DNeasy membrane. Discard the flow-through and collection tube.
It is important to dry the membrane of the DNeasy Mini spin column, since residual ethanol may interfere with subsequent reactions. This centrifugation step ensures that no residual ethanol will be carried over during the following elution. Following the centrifugation step, remove the DNeasy Mini spin column carefully so that the column does not come into contact with the flow-through, since this will result in carryover of ethanol. If carryover of ethanol occurs, empty the collection tube, then reuse it in another centrifugation for 1 min at 20,000 x g (14,000 rpm).
Place the DNeasy Mini spin column in a clean 1.5 ml or 2 ml microcentrifuge tube, and pipet 200 µl Buffer AE directly onto the DNeasy membrane. Incubate at room temperature for 1 min, and then centrifuge for 1 min at ≥6000 x g (8000 rpm) to elute.
Elution with 100 µl (instead of 200 µl) increases the final DNA concentration in the eluate, but also decreases the overall DNA yield (for details, please see DNeasy Blood & Tissue Handbook).
For maximum DNA yield, repeat elution once as described in step 13. This step leads to increased overall DNA yield. A new microcentrifuge tube can be used for the second elution step to prevent dilution of the first eluate. Alternatively, to combine the eluates, the microcentrifuge tube from step 13 can be reused for the second elution step.
Note: Do not elute more than 200 µl into a 1.5 ml microcentrifuge tube because the DNeasy Mini spin column will come into contact with the eluate.
DNA Quantification
Determine genomic DNA concentration using a Qubit Fluorometer according to the manufacturer’s instructions.
Assess DNA purity by measuring the A260/280 and A260/230 ratios using a NanoDrop spectrophotometer.