Apr 14, 2026
A Simple and Rapid Agarose Gel Electrophoresis Method to Assess CpG Methylation of DNA
- Marco Paliza-Carre1,
- Ge Chen1,
- Shahrokh Shabahang1
- 1Aditxt, Inc.
Protocol Citation: Marco Paliza-Carre, Ge Chen, Shahrokh Shabahang 2026. A Simple and Rapid Agarose Gel Electrophoresis Method to Assess CpG Methylation of DNA. protocols.io https://dx.doi.org/10.17504/protocols.io.81wgbw3nqgpk/v1
Manuscript citation:
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: November 25, 2025
Last Modified: April 14, 2026
Protocol Integer ID: 233509
Keywords: CpG methylation, agarose gel electrophoresis, methylation sensitive restriction enzyme, manufactured therapeutic DNA, cpg methylation of dna, cpg methylation level, cpg methylation, methylation level, analyzed dna, methylation pattern, manufactured therapeutic dna, using methylation, dna construct psv40, total for the analyzed dna, therapeutic dna, rapid agarose gel electrophoresis method, agarose gel electrophoresi, autoimmune disease, associated autoimmune disease, sensitive restriction enzyme digest, dna
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Abstract
This protocol describes a method for quantitative measurement of CpG methylation levels and patterns of manufactured therapeutic DNAs using methylation-sensitive restriction enzyme digest, agarose gel electrophoresis, and custom in silico band pattern analysis. The specific protocol is optimized for DNA construct pSV40-sGAD55-BLa which is a component ADI-100, an immune tolerance-inducing drug candidate for treatment of type 1 diabetes and other GAD-associated autoimmune diseases. The outputs form this procedure are values for mean Rf (mRf) and intermediate/total for the analyzed DNA, which quantitatively describe methylation level and methylation pattern respectively.
Guidelines
The selection of plasmid linearization restriction enzyme, in this case KpnI-HF, is specific to the DNA plasmid analyzed here. Select an appropriate linearization enzyme, or omit this enzyme if appropriate, for the construct being analyzed. Otherwise, the analysis parameters described in the procedure should be effective for analyzing most DNA constructs. Gel running conditions listed in this protocol are optimized for the Owl D3-14 Wide Gel Electrophoresis System. If other changes are still required, we suggest modifying MSRE selection, gel running conditions, or in silico gel analysis protocol.
Materials
Reagents
- HyPure Molecular Biology Grade Water (Cytiva #SH30538.03)
- 10x rCutSmart (New England Biolabs #B6004S)
- KpnI-HF (New England Biolabs #R3142S)
- HpaII (New England Biolabs #R0171L)
- MspI (New England Biolabs #R0106S)
- TriTrack DNA Loading Dye (6X) (Thermo Scientific #R1161)
- Sodium Dodecyl Sulfate (Certified) ACS, Fisher Chemical (Fisher Scientific #O2674-25)
- NERL™ High Purity Water (Thermo Scientific #9805)
- GeneRuler 1 kb Plus DNA Ladder (Thermo Scientific #SM1331)
- Tris-acetate EDTA (TAE) 10x solution (Fisher Scientific #BP1335-500)
- TopVision Agarose (Thermo Scientific #R0492)
- SYBR Safe (Invitrogen #S33102)
Equipment
- Pipets (P10, P100, P1000)
- Pipet Filler
- Water Bath
- Floating rack
- Microcentrifuge
- Owl D3-14 Wide Gel Electrophoresis System (Thermo Scientific #D3-14 or equivalent)
- Microwell comb (Thermo Scientific #D3-MT2D or equivalent)
- Graduated cylinder
- Axygen Gel Documentation system (Corning #GDBL-1000 or equivalent)
- TotalLab CLIQS
- Microsoft Excel
- DNase-free serological pipets
- DNase-free 1.5 mL microcentrifuge tubes
- DNase-free pipet tips
Before start
Note: This protocol is optimized for methylation analysis of pSV40-sGAD55-BLa and dbSV40-sGAD55.
The protocol may need to be optimized for use with different DNAs.
Workflow
At least three samples should be included in each run:
1. Test sample digested by KpnI/HpaII to assess methylation
2. Test sample digested by KpnI/MspI to visualize complete KpnI/HpaII digest pattern if there was no methylation
3. Hypomethylated DNA digested by KpnI/HpaII to ensure complete KpnI and HpaII digestion.
KpnI is included for plasmid linearization – this is not needed for linear DNA applications
Restriction Enzyme Digest
1h
Digest 1 µg of DNA per 50 µL reaction.
Add reagents to 1.5 mL microcentrifuge tubes as listed below.
| A | B | C | D | E | F | G | |
| Nuclease-free H20 | rCutsmart | DNA | KpnI-HF | HpaII | MspI | ||
| KpnI/HpaII digest | Volume to achieve final reaction volume of 50 µL | 5 µL | Volume equivalent to 1 µg | 1 µL | 1 µL | 0 µL | |
| KpnI/MspI digest | Volume to achieve final reaction volume of 50 µL | 5 µL | Volume equivalent to 1 µg | 1 µL | 0 µL | 1 µL |
Invert 10 times to mix.
Quick spin in microcentrifuge up to 8000 rpm/6000 g.
Incubate in 37°C water bath for 1 hr.
1h
Add 10 µL of 6x TriTrack DNA loading dye and 3.16 µL of 10% SDS to each reaction.
Agarose Gel Electrophoresis, staining, and imaging
2h 40m
Prepare a 0.5 cm thick 1.5% agarose gel.
Prepare 900 mL of 1x TAE running buffer.
Load 100 ng of sample or 300 ng of GeneRuler 1 kb+ ladder per lane. The wells should be 3 mm wide and 1.5 mm thick well. Ensure that ladders flank the sample(s).
Run gel at 100V (6.7 volts/centimeter) for 100 minutes.
1h 40m
Make a solution of 1x SYBR Safe in 150 mL 1x TAE.
Cover gel to protect from light and stain in staining solution 1 hr at 150 rpm on shaker.
1h
Image using gel documentation system with blue light. Use a 400-millisecond exposure time - this may need to be optimized if a different system is used.
Ensure that none of the bands intended for analysis are oversaturated.
Quantitation of gel bands
Open TotalLab CLIQS gel analysis software and open the gel image file.
Generate a new analysis protocol with the following settings.
Check box “Invert Image Intensity”
Rolling Ball: 200
Minimum: 1
Noise reduction: 5
%Max Peak: 1
Maximum peak of the: Gel
Edges: Automatic Detection
On the gel image, select a minimal area of interest (AOI) that contains all bands of ladders and samples.
Run the protocol you generated above.
Confirm that lanes were accurately generated, and bands were accurately defined.
Set up Rf calibration
Open the “Rf Calibration” tab.
Select keep Rf lines aligned with no lane.
Deselect “Use curved Rf lines”.
Select “Snap Rf lines to Bands when dragging”.
Drag the points from the 0 Rf line to the top 20,000 bp band of each ladder.
Drag the points from the 1 Rf line to the bottom 75 bp band of each ladder.
Delete any extra points that are not snapped to the top or bottom ladder bands and ensure that top Rf line is still labeled 0.000 and bottom Rf line is still labeled 1.000.
Calculation of mRf and intermediate/total
Select the relevant lane or “all lanes” tab at the bottom of the window.
At the top of the window, select export -> export to clipboard -> measurements table.
Paste into Microsoft Excel and use the formula below to calculate mRf, where Rf1 = Rf of band 1, V1 = raw volume of band 1, and n = the number of bands detected in the lane
Determine which bands constitute the intermediate
bands – these lie between the bands produced from digestion of
fully methylated or fully unmethylated DNA molecules. For
pSV40-sGAD55-BLa, these are bands that fall between 4880 bp and 700 bp, not including
bands at 4880 bp or 700 bp. Then use Microsoft Excel to calculate value for
intermediate/total using the formula below.