Agarose Gel Concentration Selector and Mass Calculator (with TBE/TAE Guidance)
The question of which gel concentration to run does not have a single agreed-upon answer.
For a 0.5% gel, Bio-Rad’s chart lists 1–30 kb, while miniPCR’s lists 2–50 kb; at 1.0%, Bio-Rad gives 500 bp–10 kb and miniPCR gives 400–8,000 bp. In the commonly used range (0.7–2%) sources largely agree, but at the extremes values can differ by a factor of two.
Concentration is therefore a coarse selection: it determines whether you can resolve two bands at all, not exactly where they will run.
Resolution Range (Bio-Rad Bulletin 6206)
| Concentration | Resolution range |
|---|---|
| 0.50% | 1–30 kb |
| 0.75% | 800 bp–10 kb |
| 1.00% | 500 bp–10 kb |
| 1.25% | 400 bp–7 kb |
| 1.50% | 200 bp–3 kb |
| 2.00% | 100 bp–2.5 kb |
| 3.00% | 40 bp–2 kb |
| 4.00% | 10–400 bp |
At 3% and 4%, use sieving agarose rather than standard agarose — standard agarose at these concentrations becomes brittle and difficult to pour.
Weighing
agarose (g) = concentration (%) × gel volume (mL) ÷ 100
That is: 1 g dissolved in 100 mL buffer gives 1%. No dispute there.
Volume is easy to overestimate: length × width × depth of the gel tray gives the geometric volume, but you never fill it to the brim. In Bio-Rad’s own chart, a 7 × 7 cm tray at 0.5 cm depth is listed as 20 mL, while the geometric volume is 24.5 mL.
This fill factor is not a fixed ratio: from the same Bio-Rad chart, the 7×7 tray fills to 0.82 of geometric volume, the 15×7 to 0.76, and the 15×15 to 0.89 — so there is no universal correction factor, and anyone who quotes you a fixed discount is guessing. This tool calculates from geometric volume (exact arithmetic, and it is an upper bound) and shows the manufacturer value range alongside. Weighing slightly more agarose makes the gel a bit stiffer; it will not ruin the run.
Buffer: TBE or TAE
This matters less for resolution and more for downstream steps. Per NEB:
- TBE gives better resolution for small fragments; fragments below 15 bp may not resolve in TAE. NEB recommends TBE for routine gel electrophoresis.
- Borate inhibits many enzymes. If you plan to gel-purify, ligate, digest, or sequence the band, TBE carryover can interfere; use TAE in those cases.
- For large fragments (≥ 12–15 kb) use TAE with low field strength (1–2 V/cm).
In short: use TBE if you are only visualizing; use TAE if you are cutting out the band for downstream use.
For preparing the buffer itself, see Buffer Preparation Calculator.
After the Run
To estimate fragment size from band position, see Gel Band Size — it fits a semi-log regression to your marker lanes, which is considerably more accurate than interpolating by eye.
What This Tool Does Not Cover
- Staining and imaging: ethidium bromide, GelRed, and SYBR differ in sensitivity and safety requirements; not covered here.
- Voltage and run time: depend on gel length and buffer; large fragments require low field strength and longer runs (see above).
- RNA gels: denaturing gels (formaldehyde or glyoxal) are a separate system; this tool covers only standard DNA gels.
- Pulsed-field electrophoresis: separating fragments above 30–50 kb requires PFGE; conventional electrophoresis cannot achieve this.
Resolution range table from Bio-Rad Bulletin 6206; buffer recommendations from NEB’s published guidelines.
FAQ
Why do different sources give different resolution ranges?
Because these tables are empirical — each manufacturer calibrates them against their own products and conditions. For 0.5% gels, Bio-Rad lists 1–30 kb and miniPCR lists 2–50 kb; at 1.0%, one gives 500 bp–10 kb and the other 400–8,000 bp. In the commonly used range (0.7–2%) sources largely agree; at the extremes they can differ by a factor of two. Treat concentration as a coarse selection — it determines whether two bands can separate at all, not a precise ruler.
How much agarose should I weigh out?
agarose (g) = concentration (%) × gel volume (mL) ÷ 100 — that is, 1 g in 100 mL gives 1%. **The easy mistake is the volume**: length × width × depth gives the geometric volume, but you never fill a tray to the brim. In Bio-Rad's own chart, a 7 × 7 cm tray at 0.5 cm depth is listed as 20 mL, but the geometric volume is 24.5 mL. And this fill factor **is not a fixed ratio** — from the same table, the 7×7 tray is 0.82, the 15×7 is 0.76, and the 15×15 is 0.89, so there is no universal coefficient. This tool calculates from geometric volume and shows the manufacturer range alongside; a bit of extra agarose just makes the gel slightly stiffer — it will not fail.
How do I decide between TBE and TAE?
Use TBE if you are only visualizing the band; use TAE if you are cutting out the band for downstream use. TBE gives better resolution for small fragments (below 15 bp may not resolve in TAE), and NEB recommends it for routine electrophoresis. But **borate inhibits many enzymes**, so TBE carryover after gel purification can interfere with ligation, digestion, and sequencing. For fragments above 12–15 kb, NEB recommends TAE with low field strength (1–2 V/cm).
Why are 3% and 4% gels difficult to pour?
Standard agarose at these concentrations sets brittle and tends to solidify unevenly. Use sieving agarose, which is formulated for small-fragment separation. If you only occasionally need to resolve fragments below 100 bp, polyacrylamide gels are usually easier than trying to pour 4% standard agarose.
What if my fragment size range is very wide?
One gel cannot resolve both ends. If you need to see 100 bp and 20 kb simultaneously, no single concentration covers that — each row in the table spans roughly a 10–30-fold size range, and if your range exceeds that you need two gels at different concentrations. For fragments above 30–50 kb, conventional electrophoresis cannot separate them; use pulsed-field gel electrophoresis (PFGE).
How do I estimate band size after the run?
Don't interpolate by eye between marker bands — migration distance and fragment size follow a semi-log relationship, and linear interpolation systematically overestimates. The [Gel Band Size](../gel-band-size/) tool fits a semi-log regression to your marker lanes and shows you the error that linear interpolation would have introduced.
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