NsiI (Restriction Enzyme)

REF: EG15606S

Recognition & Cleavage

Recognition site: 5′-ATGCAT-3′ (palindromic)

Sequence context:

5′ ... A T G C A^▼T ... 3′
3′ ... T_▲A C G T A ... 5′

Type: II restriction endonuclease · Common use: Routine cloning & restriction mapping

Isoschizomers: EcoT22I, Mph1103I, Zsp2I (methylation sensitivities may differ)

Specs

Optimal temperature: 37 °C
Fast digest: 5–15 min in 1× CutOne® / CutOne® Color Buffer
Heat inactivation: 80 °C · 20 min
Storage: −20 °C

Pack Components

Component	EG15606S
NsiI (20 U/µl)	25 µl (500 U)
10× CutOne® Buffer	1 ml
10× CutOne® Color Buffer	1 ml

Recommended Reaction

Buffer: 1× CutOne® or 1× CutOne® Color Buffer
Incubation: 37 °C
Typical duration: 15 min (plasmid); 15–30 min (PCR product); 30–60 min (genomic DNA)

CutOne® Color Buffer includes density reagent with red/yellow tracking dyes for direct gel loading.

Quick Protocol (Fast DNA Digestion)

20 µl (plasmid): 15 µl ddH₂O, 2 µl 10× buffer, ≤1 µg DNA, 1 µl NsiI.
30 µl (PCR product): 16 µl ddH₂O, 3 µl 10× buffer, ~0.2 µg DNA, 1 µl NsiI.
If PCR is unpurified, reduce buffer to 2 µl due to residual metal ions.
50 µl (genomic DNA): 30 µl ddH₂O, 5 µl 10× buffer, 10 µl DNA (5 µg), 5 µl NsiI.
Mix gently, quick-spin; incubate at 37 °C for the indicated time.
(Optional) Heat-inactivate at 80 °C for 20 min or load directly if Color Buffer was used.

Double / Multiple Digests

Use ~1 µl of each enzyme; keep total enzyme volume ≤10% of reaction.
If enzymes require different temperatures, start at the lower temperature, then add the second enzyme and shift higher.
Increase incubation time if total volume >20 µl.

Quality Control

Functional test: 1 µg λDNA + 1 µl NsiI (20 µl) → complete digest in 15 min at 37 °C.
Prolonged incubation / star activity: 3 h at 37 °C shows no nuclease degradation; longer incubation may yield star activity.
Ligation & recutting: After 10× over-digest, >95% ligation at 22 °C; >95% ligated fragments recut by NsiI.
Non-specific endonuclease: <10% nicked/linearized plasmid after 4 h at 37 °C.

Methylation Effects

Methylase	Effect
Dam	No effect
Dcm	No effect
CpG	No effect
EcoKI	No effect
EcoBI	Some impaired

Recognition Sites in Common DNAs

DNA	# Sites	DNA	# Sites
λDNA	14	ΦX174	0
pBR322	0	pUC57	1
pUC18/19	0	SV40	3
M13mp18/19	0	Adeno2	9

Activity in Different Buffers

Buffer	Activity
CutOne®	100%
Thermo FastDigest	100%
NEB rCutSmart™	100%
Takara QuickCut™	100%

*Activities refer to the functional test conditions above.

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NsiI

Brand: Best Enzymes
Product CAT#: EG15606S
Availability: In Stock

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