Restriction Digest Setup Calculator (Units, Glycerol Limit, and Volume Breakdown)
Do the math from the unit definition: 1 µg of DNA requires 1 unit, and a typical enzyme stock is 20 U/µL — that works out to 0.05 µL. That volume is simply not pipettable.
So in practice everyone adds 1 µL, a 20-fold excess. Enzyme amount is never the bottleneck. What actually limits you is the glycerol carried in by that 1 µL.
Three Equations
Units required = DNA amount (µg) × units per µg
Enzyme volume (µL) = units ÷ enzyme concentration (U/µL)
Final glycerol (%) = enzyme volume ÷ total reaction volume × 50%
The 50% in the last line is the glycerol content of a typical enzyme storage buffer (common value). Solving it in reverse gives the rule everyone has heard but few can derive:
Enzyme volume must not exceed 10% of the total reaction volume — because 10% × 50% = 5%, and 5% is the threshold where glycerol begins to reduce specificity and cause star activity.
Units per µg by Substrate
| Substrate | Typical value | Why |
|---|---|---|
| λ DNA / purified linear fragment | 1 U/µg | This is the definition of the unit itself |
| Supercoiled plasmid (single-cut site) | 1–20 U/µg | Supercoiled topology is harder to cut |
| Genomic DNA | 10 U/µg and up | Many sites, more contaminants; overnight incubation is typical |
These three ranges are not arbitrary: the unit definition uses λ DNA as substrate, so 1 U/µg holds only for DNA that is “as easy to cut as λ.” A plasmid’s supercoiled conformation and the total number of sites to cut both push the required units higher.
Star Activity Gives No Error Message
Excess glycerol does not cause incomplete digestion — it causes cutting at unintended sites — producing extra bands on your gel that you will likely attribute to inactivated enzyme, degraded DNA, or an incorrect site prediction. That is why this tool reports final glycerol separately: it is independent of the number of units added, yet it is the first thing to check when a digest looks wrong.
Double digests deserve special attention: glycerol from both enzymes adds up. Adding 1 µL of each to a 20 µL reaction gives 10%, right at double the limit.
How Long to Incubate
- Standard reaction: 1–4 hours.
- Genomic DNA: 4 hours to overnight.
- Before leaving anything overnight, check the enzyme’s overdigestion value: enzymes rated below 12 U/16 h are not suitable for overnight incubation; at high units over extended time, star activity risk increases.
Setting Up the Reaction
10× buffer takes up 1/10 of the total volume; the remainder is DNA, enzyme, and water. This tool calculates all four components and checks whether they fit — if your DNA is too dilute, DNA volume alone can fill the reaction, in which case you need to concentrate the DNA first or scale up the total volume.
What This Tool Does Not Cover
- Buffer compatibility. Whether two enzymes share a compatible buffer, and which supplier’s buffer to use, varies by vendor and changes over time — consult the manufacturer’s double-digest tool.
- Methylation sensitivity. Dam/Dcm methylation blocks certain enzymes; this is independent of unit amount.
- Heat-inactivation conditions. These vary by enzyme; check the product manual.
Related Tools
For cut sites and recognition sequences, see Restriction Enzyme Quick Reference; for DNA concentration from OD₂₆₀, see Nucleic Acid Concentration Calculator; for the molar ratio after ligation, see DNA Ligation Calculator.
Unit definitions, glycerol limits, and plasmid unit requirements are taken from Promega’s publicly available restriction enzyme usage guide.
FAQ
What exactly is one unit of enzyme activity?
One unit is the amount of enzyme required to **completely digest 1 µg of DNA in 1 hour** under appropriate buffer and temperature conditions, with λ DNA as the standard substrate. There is a practical consequence that rarely gets spelled out: 1 µg of DNA needs only 1 U, and a typical enzyme stock is 20 U/µL — that is 0.05 µL, which is not pipettable. So in practice everyone adds 1 µL, a 20-fold excess. Enzyme amount is never the bottleneck; glycerol is.
Why can't enzyme volume exceed 10% of the reaction?
Because enzyme storage buffer is 50% glycerol, and final glycerol above 5% causes reduced specificity — star activity — in some enzymes. 10% × 50% = 5%, which is where that rule comes from. This tool reports final glycerol directly, because what actually determines it is the **ratio of enzyme volume to reaction volume**, not the number of units added.
What does excess glycerol look like in practice?
Not incomplete digestion — **cutting at unintended sites** — producing extra bands on the gel that were not predicted. The problem is that this looks exactly like inactivated enzyme, degraded DNA, or an incorrect site prediction. Almost nobody reaches for glycerol as the first explanation. That is why it is worth calculating before you start troubleshooting.
Why does plasmid need more units than λ DNA?
The unit definition uses λ DNA as substrate, so 1 U/µg holds only for DNA that is "as easy to cut as λ." Plasmid is in a supercoiled conformation that is intrinsically harder to cut; combined with the total number of sites on a given plasmid, a single-cut supercoiled plasmid commonly requires 1–20 U/µg. Genomic DNA has many sites and more contaminants, so 10 U/µg is the starting point and overnight incubation is typical.
What should I watch out for in a double digest?
**Glycerol from both enzymes adds up.** Adding 1 µL of each enzyme to a 20 µL reaction is already 10% enzyme volume — right at double the 5% glycerol limit. Either scale up the reaction or use higher-concentration enzyme stocks. Buffer compatibility is a separate issue this tool does not cover — check the manufacturer's double-digest tool.
Can I do an overnight digest?
Standard reactions finish in 1–4 hours; genomic DNA typically takes 4 hours to overnight. If you plan to go overnight, first check the enzyme's **overdigestion value**: enzymes rated below 12 U/16 h are not recommended for overnight use, because at high units over extended time the star activity risk rises. In other words, "a longer digest is always safer" does not always hold.
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