Wetlab Calculator can help calculate formula volumes.
Reaction Setup
- Assemble reactions in thermocycler tubes. Room-temp is fine.
- Mix water and buffer before adding enzyme.
- 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.
- Make 2–5% extra master mix to account for pipetting error.
- Close tubes, flick tubes a few times to mix, and centrifuge a few seconds to recollect the liquid.
- Incubate/thermocycle according to enzyme requirements.
- For test digests, heat-inactivation is generally not necessary.
Component | Volume | Notes |
|---|---|---|
Restriction endonuclease(s) | 0.05–0.2 µL | More does not usually help, but may be necessary to be pipettable without a larger master mix. |
10× Buffer | 1 µL or 1× | See enzyme manufacturer's recommendations. |
DNA parts | 0.5–5 µL | Based on concentration, aim for 100–150 ng at least. |
Deionized Water | up to 10 µL | Enzymes ≤10% rxn vol. |
For analytical digests of plasmids, 0.75 mm thick gel combs produce the tightest bands, facilitating restriction analysis. 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.