NcoI (Restriction Enzyme)

REF: EG15550S

Recognition & Cleavage

Recognition site: 5'-CCATGG-3' (palindromic)

Cleavage pattern:

5' ... C^▼C A T G G ... 3'
3' ... G G T A C_▲C ... 5'

Type: II restriction endonuclease · Common use: Routine cloning and fast DNA digestion

Isoschizomer: Bsp19I (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	EG15550S
NcoI (20 U/µl)	30 µl (600 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
Protocol: See fast-digestion setups below (scale as needed).

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× CutOne® (or Color), ≤1 µg DNA, 1 µl NcoI.
30 µl (PCR product): 16 µl ddH₂O, 3 µl 10× CutOne®, ~0.2 µg DNA, 1 µl NcoI.
If PCR is unpurified, reduce buffer to 2 µl due to residual metal ions.
50 µl (genomic DNA): 30 µl ddH₂O, 5 µl 10× CutOne®, 5 µg DNA (10 µl), 5 µl NcoI.
Mix gently, quick-spin; incubate at 37 °C for 15 min (plasmid), 15–30 min (PCR), or 30–60 min (genomic).
(Optional) Cleanup (phenol/chloroform or column). If using Color Buffer, load directly on gel.

Multiple Digests

Use 1 µl of each enzyme; keep total enzyme volume ≤10% of reaction.
If temps differ, start at the lower temperature, then add the second enzyme and shift higher.

Quality Control

Functional test: 1 µg λDNA + 1 µl NcoI in 20 µl CutOne® → complete digest in 15 min at 37 °C.
Prolonged incubation/star activity: 3 h at 37 °C shows no nuclease degradation; longer may lead to star activity.
Ligation & recutting: After 10× over-digest, >95% ligation at 22 °C; >95% of ligated fragments recut by NcoI.
Non-specific endonuclease: <10% nicked/linearized plasmid after 4 h at 37 °C.
Blue/White assay: <1% white colonies (DNA ends remain intact).

Methylation Effects

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

Recognition Sites in Common DNAs

DNA	# Sites	DNA	# Sites
λDNA	4	ΦX174	0
pBR322	0	pUC57	0
pUC18/19	0	SV40	3
M13mp18/19	0	Adeno2	20

Activity in Different Buffers

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

*Activities per functional test conditions.

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NcoI

Brand: Best Enzymes
Product CAT#: EG15550S
Availability: In Stock

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