SpeI
A▾CTAGT5′ overhang (4)
SpeI is a 6 bp-recognition restriction enzyme. It runs best in rCutSmart at 37°C, and is prone to star activity under non-standard conditions.
NEB buffer activity
| Buffer | % Activity |
|---|---|
| rNEBuffer 1.1 | 75% |
| rNEBuffer 2.1 | 100% |
| rNEBuffer 3.1 | 25% |
| rCutSmartbest | 100% |
- Incubation
- 37°C
- Heat inactivation
- 80°C, 20 min
- Star activity
- Yes
- Methylation blocked
- Dam
ACTAGT. Dam site (GATC) can overlap when flanked -> blocked in context. Some star propensity.
Find SpeI sites in your own sequence
Paste a sequence below and this scans it for every SpeI recognition site — the same engine behind the full Restriction Sites tool, scoped to just this one enzyme.
Frequently asked questions
What does SpeI recognize?
SpeI recognizes the 6 bp sequence ACTAGT (5′→3′), cutting the top strand between position 1 and 2 within the site to leave a 5′ overhang of 4 bases.
What buffer should I use for SpeI?
rCutSmart gives the highest activity (incubate at 37°C). Supplied NEBuffer 4 + BSA == rCutSmart.
Does SpeI have star activity?
Yes — under non-standard conditions (excess enzyme/glycerol, long incubation, low ionic strength) it can cleave at sites resembling but not matching ACTAGT.
Is SpeI blocked by DNA methylation?
Yes — blocked by Dam methylation. Use methylation-free template (e.g. a dam⁻/dcm⁻ strain) if this matters for your digest.
Can I heat-inactivate SpeI?
Yes — 80°C for 20 minutes.