Libre Biotech
Dashboard Projects Labbook Protocols Training Groups Sign in

EZ DNA Methylation‑Gold™ Kit — Bisulfite Conversion of DNA

### Purpose To convert unmethylated cytosines in DNA to uracil (while preserving 5‑methylcytosine) using the EZ DNA Methylation‑Gold™ chemistry for downstream PCR/sequencing or other analyses. Typical performance: **>99% conversion of unmethylated C** and **>99% protection of methylated C**; **>75% DNA recovery**. Recommended DNA input **200–500 ng** (acceptable range **500 pg–2 µg**). ### Scope For purified double‑stranded DNA from cells, tissues, plasma/serum DNA, etc., processed in 0.2 mL PCR tubes with a thermal cycler and standard microcentrifuge. The kit consolidates **heat denaturation + bisulfite conversion** and includes **in‑column desulphonation** for rapid cleanup.

sample_prep
Version History
Version 1 Current
Effective: 2025-08-26

First version.

Procedure Steps (Version 1)

Materials

Kit contents (store at room temperature)

  • CT Conversion Reagent (solid; reconstitute before use)
  • M‑Dilution Buffer
  • M‑Dissolving Buffer
  • M‑Binding Buffer
  • M‑Wash Buffer (concentrate) — add ethanol before first use
  • M‑Desulphonation Buffer
  • M‑Elution Buffer
  • Zymo‑Spin™ IC Columns with collection tubes
  • Instruction manual Refer to the table on page 1 for volumes (50‑reaction D5005 vs 200‑reaction D5006).

Additional supplies & equipment (not provided)

  • Thermal cycler with heated lid; microcentrifuge (≥10,000 × g)
  • Calibrated pipettes and aerosol‑resistant tips; 1.5 mL microfuge tubes
  • Nuclease‑free water; 100% ethanol (for M‑Wash Buffer)

Reagent preparation

CT Conversion Reagent (prepare per tube; 10 reactions/tube)

  1. Add 900 µL water + 300 µL M‑Dilution Buffer + 50 µL M‑Dissolving Buffer to one tube of CT Conversion Reagent.
  2. Mix at room temp with frequent vortexing/shaking for 10 min. (Trace undissolved material is acceptable.)
  3. Storage (light‑protected): use immediately for best results; otherwise RT overnight, 4 °C 1 week, or –20 °C up to 1 month. Before reuse, warm to 37 °C and vortex.

When using >20 µL DNA per reaction (up to 50 µL): decrease the water used to reconstitute CT Conversion Reagent by 100 µL for each +10 µL increase in DNA volume; and decrease the CT Conversion Reagent added to the sample by the same increment to keep the reaction at 150 µL (see §8.1 & table). Do not change M‑Dilution or M‑Dissolving volumes.

M‑Wash Buffer

Before first use, add 24 mL 100% ethanol to 6 mL concentrate (D5005) or 96 mL ethanol to 24 mL concentrate (D5006). Mark the bottle.

Sample requirements

  • DNA input: 500 pg–2 µg; optimal 200–500 ng per conversion. Very high inputs (near 2 µg) may reduce conversion at extremely GC‑rich loci.

Set up bisulfite conversion (per sample)

  1. In a PCR tube, combine 20 µL DNA (top up with water if <20 µL) with 130 µL CT Conversion Reagent. Mix by flicking or pipetting; quick‑spin. Final reaction volume = 150 µL.

If DNA volume >20 µL (max 50 µL): adjust as follows to maintain 150 µL total and correct CT formulation.

DNA volume (µL) CT Conversion Reagent added to sample (µL) Water used in CT reagent reconstitution (per tube)
20 130 900 µL
30 120 800 µL
40 110 700 µL
50 100 600 µL

  1. Thermal‑cycle (lid heated):

    • 98 °C 10 min → 64 °C 2.5 h → 4 °C hold (≤20 h) (hold optional).

Alternative cycling (for longer amplicons; may risk incomplete conversion for >80% GC templates): Alt‑1: 98 °C 10 min → 53 °C 30 min → (repeat 8×) [53 °C 6 min → 37 °C 30 min] → 4 °C hold. Alt‑2: 98 °C 10 min → 53 °C 4 h → 4 °C hold.

Bind DNA

  1. Add 600 µL M‑Binding Buffer to a Zymo‑Spin™ IC Column seated in a collection tube. (Capacity of tube+column = 800 µL—empty as needed.)
  2. Load the entire conversion reaction onto the column containing the M‑Binding Buffer, close cap, invert several times to mix.
  3. Centrifuge 30 s at ≥10,000 × g; discard flow‑through.

Wash & desulphonate

  1. Add 100 µL M‑Wash Buffer; spin 30 s.
  2. Add 200 µL M‑Desulphonation Buffer; stand 15–20 min at 20–30 °C, then spin 30 s.
  3. Add 200 µL M‑Wash Buffer; spin 30 s. Repeat with another 200 µL; spin 30 s.

Elute DNA

  1. Move the column to a clean 1.5 mL tube. Add 10 µL M‑Elution Buffer directly to the matrix; spin 30 s at full speed to elute. (Water or TE, pH ≥ 6.0, is acceptable for elution if required.)

Storage & use: DNA is ready for analysis or store ≤–20 °C (long‑term ≤–70 °C). For PCR, use 1–4 µL per reaction (up to 10 µL if needed). Larger elution volumes are possible but will dilute the DNA.

Quality control & acceptance criteria

  • Record that cycling followed §8.1 and all column steps in §8.2–8.4 were completed.
  • Expected performance: >99% conversion of unmethylated C; >99% protection of methylated C; >75% DNA recovery.
  • If low yield or inconsistent results: verify ethanol added to M‑Wash, correct reaction volume (150 µL) and incubation times, and that the CT reagent was prepared and stored per requirements.

Notes for downstream PCR/sequencing (best practice)

  • Minimum DNA for successful PCR from human/mouse templates ≈ 100 pg; recommended 200–500 ng input for conversion. Amplicon size 150–300 bp is most robust; 35–40 cycles typical; 55–60 °C annealing often works well. Use a hot‑start polymerase optimized for bisulfite DNA (e.g., ZymoTaq™).
  • Primer design: Design 26–32 nt primers to the converted sequence; treat C as T (except CpG sites). If CpG methylation is unknown, use a mixed base (C/T or G/A) but avoid mixed bases at the 3′ end (≤1 mixed position per primer, preferably near 5′). Example and guidance on page 11.

Primary source: EZ DNA Methylation‑Gold™ Kit — Instruction Manual, Ver. 2.1.5 (Revised 7/28/2021). All conditions, volumes, and notes above are drawn directly from the manufacturer’s manual and page‑referenced where applicable.