Wetlab Calculator can help calculate formula volumes.

Reaction Setup

Use a larger reaction with more DNA and enzyme for a preparatory digest, one where the product is to be column- or gel-purified. For restriction analysis, less DNA and enzyme can be spared

  1. Assemble reactions in thermocycler tubes. Room-temp is fine.
    1. Mix water and buffer before adding enzyme. 
    2. Make master mixes when possible, as it reduces pipetting steps, reduces errors from pipetting small volumes, and maximizes component precision across reactions. Combine all common components for n reactions, and aliquot volumes reduced by the volume of the variable components, which are generally one or more DNA parts. Wetlab Calculator.
    3. Make 2–5% extra master mix to account for pipetting error.
  2. Close tubes, flick tubes a few times to mix, and centrifuge a few seconds to recollect the liquid.
  3. Incubate/thermocycle according to enzyme requirements.
    1. For digests, heat-inactivation is generally not necessary. For preparatory digests, heat inactivation can be skipped if purifying, or if residual enzyme activity will not affect the assembly the digest will be used in.


Component

Volume,
Analytical

Volume,
Preparatory

Notes

Restriction endonuclease(s)

0.05–0.2 µL

1 µL

More does not usually help, but may be necessary to be pipettable without a larger master mix.

10× Buffer

1 µL or 1×

1×

See enzyme manufacturer's recommendations.

DNA parts

0.5–5 µL

0.5–1 µg

Based on concentration, aim for ≥100–150 ng for restriction analysis.

Deionized Water

up to 10 µL

up to 40 µL

Enzymes ≤10% rxn vol.
Aim for DNA being <½ the reaction.
Rxns master mixes can be split for even smaller volumes.

Analytical Gel Electrophoresis

For analytical digests of plasmids, 0.75 mm thick gel combs produce the tightest bands, facilitating band resolution. Each 0.75 mm thick gel well holds ≈9 µL, allowing for 7 µL digest reaction mixed with 1.4 µL loading dye. 7 µL test digests are thus Shyam's routine method.

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