DNA/RNA (and Protein) Electrophoresis
Recommended conditions for non-denaturing agarose (or PAGE) electrophoresis and practical RNA integrity assessment.
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Recommended conditions
Gel and buffer
- Use 1× THE buffer (RNA/DNA is stable during electrophoresis in these conditions)
- Agarose concentration: 1.0–1.5% for typical DNA/RNA fragments
- Stain: ethidium bromide (EtBr) or an alternative DNA stain
Safety
- Wear gloves and avoid direct skin contact with EtBr
- Dispose of gels and buffers according to local safety rules
- Prefer safer stains if required by your institution
RNA integrity assessment (non-denaturing gel)
The first sign of RNA degradation is a slight smear originating from rRNA bands and extending toward shorter fragments.
- Mammalian total RNA: two strong bands (~4.5 and ~1.9 kb; 28S and 18S rRNA) with ratio ~1.5–2.5:1.
- Non-mammalian sources (plants/insects/yeast/amphibians): typical mRNA smear may not exceed ~2–3 kb.
- Hidden break: many invertebrates have a “hidden break” in 28S rRNA; appearance may be a single 18S-like band.
Loading buffer (10×)
Recipe: 20% (w/w) Polysucrose 400 (P7798), 100 mM Tris-HCl (pH 8.0), 5 mM EDTA, ~0.01% Orange G, ~0.01% Xylene Cyanol FF.
Electrophoresis buffers
10× THE
(24.23 g Tris-base, 47.66 g HEPES (free acid), 10 ml 0.5 M EDTA; to 1 L)
10× TME (MOPS)
(24.23 g Tris-base, 44.85 g MOPS (free acid), 10 ml 0.5 M EDTA; to 1 L)
10× TME (MES)
(24.23 g Tris-base, 39.04 g MES (free acid), 10 ml 0.5 M EDTA; to 1 L)
| Tris-base (mM) | HEPES-acid (mM) | Tris/HEPES ratio | pH (20°C) |
|---|---|---|---|
| 350 | 50 | 7/1 | 9.08 |
| 300 | 50 | 6/1 | 9.01 |
| 250 | 50 | 5/1 | 8.93 |
| 200 | 50 | 4/1 | 8.82 |
| 150 | 50 | 3/1 | 8.68 |
| 100 | 50 | 2/1 | 8.46 |
| 50 | 50 | 1 | 8.06 |
| 50 | 100 | 1/2 | 7.56 |
| 50 | 150 | 1/3 | 7.30 |
| 50 | 200 | 1/4 | 7.13 |
| 50 | 250 | 1/5 | 7.00 |
| 50 | 300 | 1/6 | 6.90 |
| 50 | 350 | 1/7 | 6.80 |
| Tris-base (mM) | MOPS-acid (mM) | Tris/MOPS ratio | pH (20°C) |
|---|---|---|---|
| 60 | 20 | 3/1 | 8.74 |
| 50 | 20 | 5/2 | 8.62 |
| 40 | 20 | 2/1 | 8.46 |
| 30 | 20 | 3/2 | 8.22 |
| 20 | 20 | 1 | 7.83 |
| 20 | 30 | 2/3 | 7.43 |
| 20 | 40 | 1/2 | 7.18 |
| 20 | 50 | 2/5 | 7.01 |
| 20 | 60 | 1/3 | 6.89 |
| Tris-base (mM) | MES-acid (mM) | Tris/MES ratio | pH (20°C) |
|---|---|---|---|
| 60 | 20 | 3/1 | 8.76 |
| 50 | 20 | 5/2 | 8.64 |
| 40 | 20 | 2/1 | 8.47 |
| 30 | 20 | 3/2 | 8.19 |
| 20 | 20 | 1 | 7.50 |
| 20 | 30 | 2/3 | 6.56 |
| 20 | 40 | 1/2 | 6.22 |
| 20 | 50 | 2/5 | 6.02 |
| 20 | 60 | 1/3 | 5.88 |
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