Type II restriction enzymes in practice: bsmi and the incubation temperature that catches people out, and choosing an isoschizomer when a site is blocked
BsmI is one of the restriction enzymes whose optimum incubation sits above the temperature most people set a digest at out of habit, and running it at the wrong temperature is the single most common reason a BsmI digest disappoints. Around that sit the usual questions of unit definition, buffer compatibility and methylation blocking, plus the useful option of substituting an isoschizomer. This page covers all of them.
- the FDA labelling clause behind research use only on a reagent
- 809.10(c)
- good laboratory practice for nonclinical studies, 21 CFR
- Part 58
- hazard communication, which decides what the container must tell the user
- 1910.1200
Figures in this panel are the rules that decide what a reagent may claim and what its container must say, named from the regulations themselves and linked in the sources below. They are identifiers, not prices: BioBricks publishes verified prices for synthesis services only, and does not imply a reagent price index it has not measured.
- 4 vendor service pages verifiedevery figure matched verbatim to the vendor's page
- Quoted and dated, never estimatedlast verification pass 2026-08-24
- 1 service classes coveredeach with measured search demand behind it
Getting the digest right
- Use the supplier's stated temperature. Several type II enzymes including this one have an elevated optimum, and a digest set at the conventional temperature will run slowly or incompletely. Read the data sheet rather than assuming, and check the block or bath actually holds the set point.
- Check thermal inactivation separately. Whether an enzyme can be heat inactivated, and at what temperature, is a separate property from its incubation optimum. An enzyme that cannot be inactivated by heat needs a column clean-up or a spin before the next step, and that changes the protocol timing.
- Methylation can block the site. Overlapping host methylation blocks some sites entirely, and whether yours is affected depends on the strain the DNA came from. Where it is, growing the plasmid in a methylation deficient strain is usually simpler than changing the strategy.
- Isoschizomers and neoschizomers. Another enzyme recognising the same sequence may cut where this one cannot, or work in a buffer that suits your double digest better. A neoschizomer cuts the same site at a different position, which changes the overhang, so check the cut position and not only the recognition sequence.
- Confirm the site before you order. Run the sequence against a restriction analysis tool and confirm the number and position of sites in the actual construct, including the vector. Ordering an enzyme for a site that the vector also carries is a wasted week.
When to substitute rather than troubleshoot
If a site is methylation blocked, the buffer is incompatible with the partner enzyme, or the incubation temperature is inconvenient for the workflow, substituting an isoschizomer is usually faster than optimising. Suppliers list them, and the substitution is a data sheet comparison rather than an experiment.
Check the overhang the substitute leaves. A neoschizomer that cuts at a different position within the same recognition sequence produces an end that will not ligate with what you planned.
Buying sensibly
Enzymes used occasionally should be bought in the smallest useful size, because activity declines with storage and handling and a large tube used twice a year is mostly waste. Enzymes used constantly are worth buying in bulk and aliquoting once.
Keep the lot and the data sheet with the protocol. Supplier buffer formulations and recommended conditions do change between catalogue revisions, and a protocol that cites a buffer by name without a date can quietly become wrong.
Common questions
- What temperature should a BsmI digest run at?
- Above the conventional digest temperature: check the supplier's data sheet for the stated optimum and set the block to it. Running it at the habitual temperature is the most common cause of an incomplete digest with this enzyme.
- What is an isoschizomer?
- A different enzyme recognising the same sequence. It may cut where the original is blocked by methylation or work in a more convenient buffer. A neoschizomer recognises the same sequence but cuts at a different position, changing the overhang.
- Can BsmI be heat inactivated?
- Check the data sheet, because inactivation temperature is a separate property from incubation temperature and not every enzyme can be inactivated by heat. Where it cannot, clean up before the next enzymatic step.
- Why does my construct have unexpected sites?
- The vector usually carries sites too. Run a restriction analysis over the complete construct including the backbone before ordering, rather than over the insert alone.
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The median advertised gene synthesis price per base pair in the US research synthesis services market was $0.11 in August 2026, across 4 verified vendor service pages recorded in BioBricks Synthesis Price Index.
Cite as: "BioBricks Synthesis Price Index", updated 2026-08-24, https://biobricks.org/type-ii-restriction-enzymes/.