BioDeviceHub

Restriction Digest Reaction Setup

Plan a restriction digest reaction: the volumes of DNA, buffer, each enzyme and water for a chosen DNA amount, units per microgram and total volume, with a glycerol check.

Formula

VDNA=mDNACDNAV_{\mathrm{DNA}} = \dfrac{m_{\mathrm{DNA}}}{C_{\mathrm{DNA}}}
Venzyme=(U/μg)×mDNA (μg)stock (U/μL)V_{\text{enzyme}} = \dfrac{(\mathrm{U}/\mu\mathrm{g})\times m_{\mathrm{DNA}}\,(\mu\mathrm{g})}{\mathrm{stock}\ (\mathrm{U}/\mu\mathrm{L})}
Vwater=Vtotal−VDNA−Vbuffer−VenzymesV_{\text{water}} = V_{\text{total}} - V_{\mathrm{DNA}} - V_{\text{buffer}} - V_{\text{enzymes}}
U\mathrm{U}
the amount of enzyme that digests 1 µg of DNA in 1 hour under the supplier's conditions
stock\text{stock}
enzyme concentration printed on the tube, in units/µL

How it works

A digest is a small reaction: DNA, a buffer at one-tenth of the final volume, the enzyme and water. The enzyme is defined in units, where one unit digests 1 µg of a standard DNA in an hour, so the number of units follows from the amount of DNA and how thoroughly or quickly you need it cut. A few units per microgram is a common start; more is used for short digests or difficult DNA.

Enzymes are supplied in glycerol, and too much glycerol in the reaction makes many enzymes cut at related but wrong sites (star activity). Keeping the enzymes to no more than a tenth of the reaction volume keeps the glycerol near or below 5%. The tool warns when the enzymes exceed that share.

Worked example

1 µg of DNA at 200 ng/µL, two enzymes at 20 units/µL, 5 units of each per µg, in 50 µL with a 10× buffer.

  1. DNA = 1000 ng / 200 ng/µL = 5.0 µL.
  2. Each enzyme = 5 × 1 / 20 = 0.25 µL.
  3. Buffer = 50 / 10 = 5.0 µL.
  4. Water = 50 − 5.0 − 5.0 − 0.5 = 39.5 µL.

Combine 39.5 µL of water, 5 µL of buffer, 5 µL of DNA and 0.25 µL of each enzyme. The enzymes are 1% of the reaction.

These are the values the calculator opens with, so you can check its output against this example.

Assumptions

  • The enzyme concentration on the tube is correct for the lot in use.
  • All enzymes work in the buffer you choose, at the incubation temperature.
  • The DNA is free of contaminants that inhibit digestion, such as salts, ethanol or phenol.

Common mistakes

  • Pipetting 0.25 µL directly. Dilute the enzyme in its storage buffer or make a master mix for several reactions.
  • Adding enzyme to cold reagents before the buffer, which can precipitate some enzymes.
  • Carrying a large volume of DNA in TE buffer into the reaction, where the EDTA takes up magnesium the enzyme needs.

Related tools

Restriction Digest PredictorFragment sizes for a single or multiple enzyme digestOpen calculatorRestriction Site FinderSites, cut positions and overhangs for named restriction enzymesOpen calculatorLigation Calculator (Vector:Insert Ratio)Insert mass for a molar ratio, or the ratio from the masses you haveOpen calculatorMaster Mix CalculatorScale any reaction recipe to a batch, with overageOpen calculator

Related equipment

Service documentation, failure modes and parts for the instruments this calculation is used with.