Agarose Gel Electrophoresis for DNA Integrity/Size Assessment

Safety

• Wear lab coat, gloves, and eye protection.
• If using ethidium bromide (EtBr), treat gel and buffer waste as hazardous. Prefer SYBR‑Safe or equivalent when available.
• Use UV/blue‑light transilluminators with shielding.

Materials & Equipment

• Agarose powder

• TAE buffer (1×): 40 mM Tris‑acetate, 1 mM EDTA

• DNA stain: SYBR‑Safe, GelRed, or EtBr (0.5 µg/mL if in‑gel)

• Loading dye (6×) with tracking dyes and glycerol

• DNA ladders (or equivalents):

  • 100‑bp ladder (e.g., Bioline HyperLadder IV)
  • 1‑kb ladder

• Nuclease‑free water

• Microwave or hot plate; gel tray/comb; electrophoresis tank & power supply

• Pipettes & wide‑bore tips (recommended for HMW DNA)

• Imaging system (blue light or UV)

Definitions

• HMW DNA: >30–50 kb; typically migrates poorly in standard agarose and may remain near the wells.

Gel preparation (0.75% agarose in 1× TAE)

1. For 100 mL gel, combine 0.75 g agarose with 100 mL 1× TAE in a microwave‑safe flask.
2. Heat until fully dissolved; swirl to clear.
3. Cool to ~60 °C. Add stain if using in‑gel (per manufacturer).
4. Pour into casting tray with suitable comb(s). Allow to solidify (≥30 min).
5. Place gel in tank and cover with 1× TAE so gel is submerged by 2–3 mm.

Note: 0.75% is optimal for showing whether gDNA is intact. For finer resolution of 0.2–3 kb products, use 1.0–2.0% agarose.

Sample preparation

1. Determine DNA concentration (e.g., Qubit/NanoDrop).
2. Target 50–150 ng DNA per lane for QC. For viscous HMW DNA, avoid >200 ng to reduce “plugging” in the well.
3. Mix 5 volumes DNA with 1 volume 6× loading dye to achieve 1× final.
4. If samples are very viscous, pre‑dilute 1:5–1:10 in TE or water to aid entry into the gel.
5. Optional: RNase treatment if RNA contamination is suspected.

Electrophoresis

1. Flush wells with buffer if needed. Carefully load ladders (3–5 µL) and prepared samples.

2. Run conditions (default):

   • 90 V for 45 min, then 120 V for 30 min (total ~75 min).
   • Confirm typical current (record V and mA).

3. Stop the run when the faster tracking dye has migrated ~60–75% of gel length.

Alternative (recommended for HMW DNA to reduce “smiling”): constant 70–90 V for 90–120 min.

Imaging & documentation

1. Transfer gel to imager (or image in tank if safe).
2. Capture high‑resolution images with exposure that keeps ladder bands unsaturated.
3. Save files as TIFF (archival) and JPEG/PNG (reporting).
4. Annotate the image with date, gel %, buffer, ladder types, run conditions, lane map, and operator initials.

Ladder quality check

• Bands should be sharp and straight in both end ladders.
• Pronounced curvature (“smiling”) or compression at the top suggests excessive voltage/heat.

Sample assessment

• High‑quality gDNA: Bright HMW material near/just below the wells, with a light tapering smear into the 10–50 kb region; minimal signal below ~2 kb.
• Overloaded/very large DNA: Large fraction remains in the well. Mitigate by loading less or diluting (Section 9).
• Degradation: Diffuse smear extending broadly into low sizes (<2 kb) or a dominant low‑molecular‑weight hump.
• RNA contamination: Hazy, fast‑migrating smear near 100–300 bp; treat with RNase and/or clean up.
• Salt/contaminants: Distorted lanes, DNA reluctant to enter gel, or fuzzy bands; perform cleanup.
• PCR products/restriction digests: Expect discrete bands; unexpected extra bands or smears indicate non‑specific amplification or partial digestion.

Pass/flag criteria (record outcome)

• Pass (gDNA for most library preps): HMW plug with minimal low‑MW smear.
• Flag/Investigate: weak lanes (low yield), heavy low‑MW smear, or strong well retention that persists after dilution.
• Fail: extensive degradation (<1 kb smear predominant) or no visible DNA at reasonable loading mass.

Troubleshooting

Observation → Likely cause → Corrective action

• DNA remains in/near wells → very large fragments and/or overloaded; salt carryover; high voltage heat → Load 50–100 ng, dilute viscous samples 1:5–1:10; perform bead/EtOH cleanup; run at constant 70–80 V and keep buffer cool.
• Faint sample lane → under‑loaded or low extraction yield → quantify accurately; increase mass or concentrate; verify loading.
• Smiling/curved bands → overheating/high voltage; uneven buffer depth → reduce voltage, extend time; ensure even gel/buffer; avoid placing gel too close to electrodes.
• Diffuse bands → old buffer, contaminated agarose, or too much intercalating dye → replace buffer/gel; use recommended dye concentration.
• Abundant low‑MW haze → RNA → RNase A treat (e.g., 10–20 µg/mL, 37 °C 15–30 min) and re‑run.

Appendices

A. Quick recipe table

• 1× TAE (1 L): 40 mM Tris‑acetate, 1 mM EDTA (use commercial 50× or prepare stock and dilute).
• 0.75% gel (100 mL): 0.75 g agarose + 100 mL 1× TAE.
• Loading dye to 1×: mix 5 parts sample : 1 part 6× dye (e.g., 8 µL DNA + 2 µL dye).

B. Optional controls

• Dilution control: load a 1:5 dilution of a viscous sample to distinguish overload from degradation.
• Digest control (gDNA): digest 100–200 ng with a frequent cutter; a broad 0.5–20 kb smear indicates intact DNA.

Notes:

• This SOP’s default (0.75% TAE, 90 V → 120 V) reproduces the conditions under which HMW DNA commonly appears as a bright plug near the wells. Adjust gel % and voltage/time to match the size range and resolution you need.
• For sizing >50–100 kb accurately, use PFGE or capillary systems (e.g., Femto Pulse); standard agarose gels only provide a coarse integrity check for very large DNA.