Agarose gel calculator
Grams of agarose, buffer and stain for any percentage and volume, and which percentage to choose.
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Grams of agarose, buffer and stain for any percentage and volume, and which percentage to choose.
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A gel percentage is weight per volume, so a 1 % gel has 1 g in 100 mL. For a 50 mL minigel at 1 % that is 0.5 g; for a 150 mL gel at 2 % it is 3 g. The volume is the tray's length times width times the depth you pour, in centimetres, which gives millilitres directly. Prepare a little extra: some buffer evaporates while heating, and it is better to discard a few millilitres than to pour a gel that does not reach the comb.
Agarose forms a sieve, and its pore size shrinks as the percentage rises. Large DNA needs big pores and a low percentage; small fragments need fine pores and a high percentage. Pick the percentage whose range brackets the fragments you need to tell apart.
| Agarose | Best for | Typical use |
|---|---|---|
| 0.5 % | 1–30 kb | Genomic DNA, BACs, very large fragments; fragile |
| 0.7 % | 0.8–12 kb | Large plasmids, lambda digests |
| 1.0 % | 0.5–10 kb | Plasmids, most PCR products, restriction digests |
| 1.2 % | 0.4–7 kb | General purpose with slightly better small-fragment resolution |
| 1.5 % | 0.2–3 kb | Small PCR products, genotyping |
| 2.0 % | 50 bp–2 kb | Short amplicons, small digests |
| 3.0 % | 10 bp–1 kb | Primers, oligos, small differences; use a high-resolution agarose |
Predict your fragment sizes with the PCR product size calculatoror the restriction digest tool, and check a sequence's length with the DNA length calculator.
TAE (Tris-acetate-EDTA) has a lower buffering capacity, so it exhausts on long runs, but DNA migrates faster in it, large fragments resolve better, and DNA is recovered from it more easily, which makes it the choice for preparative gels and cloning. TBE(Tris-borate-EDTA) buffers better, gives sharper bands for fragments under about 2 kb, and suits long or high-voltage runs, but borate inhibits some enzymes and complicates gel extraction. Whichever you use, make the gel in the same buffer you run it in; a gel cast in TBE and run in TAE gives smeared bands.
Ethidium bromide is the classic stain, used at 0.5 µg/mL, 5 µL of a 10 mg/mL stock per 100 mL. It intercalates DNA and is a mutagen, so handle it with gloves and dispose of gels as hazardous waste. SYBR Safe and GelRed are used at 1:10,000 and are far less hazardous; GelRed is best added to the gel, SYBR Safe works both in-gel and as a post-stain. Adding any stain to hot agarose degrades it, so wait until the solution is about 55 °C.
1 g of agarose for every 100 mL of buffer. A 1% gel is 1% w/v, so a 50 mL minigel needs 0.5 g and a 150 mL gel needs 1.5 g. Weigh the agarose, add the buffer, and heat until fully dissolved.
Lower percentages separate large fragments and higher percentages separate small ones. Use 0.7% for fragments over 5 kb, 1% for the usual 0.5–10 kb range such as plasmids and PCR products, 1.5–2% for fragments under 1 kb, and 3% or a special high-resolution agarose for anything under 200 bp.
TAE for preparative work and large fragments, because it has lower buffering capacity but DNA recovers more easily from it and large DNA migrates better. TBE for small fragments and long runs, because it buffers better and gives sharper bands below about 2 kb. Make the gel and run it in the same buffer.
To a final concentration of 0.5 µg/mL: 5 µL of the standard 10 mg/mL stock per 100 mL of gel. Add it once the melted agarose has cooled to about 55 °C, and remember it is a mutagen. SYBR Safe and GelRed are used at 1:10,000, 10 µL per 100 mL, and are safer alternatives.
Usually because the agarose was not fully dissolved: look for floating clear lumps before pouring and keep heating until the solution is completely clear. Pouring when too hot warps trays and evaporates buffer, which raises the percentage; pouring when too cool gives an uneven gel. Aim for about 55 °C, warm to the touch.