XhoI (Restriction Enzyme)

REF: EG15583S

Recognition & Cleavage

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

Cleavage pattern:

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

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

Isoschizomers: PaeR7I, Sfr274I, SlaI (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	EG15583S
XhoI (20 U/µl)	500 µl (10,000 U)
10× CutOne® Buffer	3 × 1 ml
10× CutOne® Color Buffer	3 × 1 ml

Recommended Reaction

Buffer: 1× CutOne® or 1× CutOne® Color Buffer
Incubation: 37 °C
Typical duration: 5–15 min (plasmid); 15–30 min (PCR); 30–60 min (genomic DNA)

CutOne® Color Buffer contains density reagent + tracking dyes for direct gel loading (red ≈2.5 kb; yellow ≈10 bp in 1% agarose).

Quick Protocol (Fast DNA Digestion)

20 µl (plasmid): 15 µl ddH₂O, 2 µl 10× CutOne®, ≤1 µg DNA, 1 µl XhoI.
30 µl (PCR product): 16 µl ddH₂O, 3 µl 10× CutOne®, ~0.2 µg DNA, 1 µl XhoI.
If PCR not purified, use 2 µl 10× buffer.
50 µl (genomic): 30 µl ddH₂O, 5 µl 10× CutOne®, 10 µl DNA (5 µg), 5 µl XhoI.
Mix gently, quick-spin; incubate at 37 °C for the indicated time.
(Optional) Heat-inactivate at 80 °C for 20 min or load gel directly (Color Buffer).

Notes

Total enzyme volume ≤10% of reaction (limit glycerol to avoid star activity).
Contaminants (phenol, ethanol, EDTA, high salt, detergents) inhibit digestion—purify DNA if needed.
Increase incubation time if total volume >20 µl.

Quality Control

Functional test: Complete λDNA digest (HindIII digest substrate) in 15 min at 37 °C (20 µl, 1 µg DNA, 1 µl enzyme).
Prolonged incubation/star activity: 3 h at 37 °C shows no nuclease degradation; longer incubation may result in star activity.
Ligation & recutting: >95% ligation at 22 °C with T4 DNA ligase; >95% of ligated fragments recut by XhoI.
Non-specific endonuclease: <10% nicked/linearized 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	Impaired
EcoKI	No effect
EcoBI	No effect

Recognition Sites in Common DNAs

DNA	# Sites	DNA	# Sites
λDNA	1	ΦX174	1
pBR322	0	pUC57	0
pUC18/19	0	SV40	0
M13mp18/19	0	Adeno2	6

Activity in Different Buffers

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

*Activities per the functional test conditions.

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XhoI

Brand: Best Enzymes
Product CAT#: EG15583S
Availability: In Stock

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