MluI (Restriction Enzyme)

REF: EG15547S

Recognition & Cleavage

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

Sequence context:

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

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

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	EG15547S
MluI (20 U/µl)	100 µl (2000 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
Reference: See fast-digestion protocol below; scale volumes as needed.

CutOne® Color Buffer includes density reagent and 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 MluI.
30 µl (PCR product): 16 µl ddH₂O, 3 µl 10× CutOne®, ~0.2 µg DNA, 1 µl MluI.
If PCR is unpurified, reduce buffer to 2 µl due to residual metal ions.
50 µl (genomic): 30 µl ddH₂O, 5 µl 10× CutOne®, 10 µl DNA (5 µg), 5 µl MluI.
Mix gently, quick-spin; incubate at 37 °C for 15 min (plasmid), 15–30 min (PCR), or 30–60 min (genomic).
(Optional) Heat-inactivate at 80 °C for 20 min or load gel directly if Color Buffer was used.

Multiple Digests

Use ~1 µl of each enzyme; keep total enzyme volume ≤10% of reaction.
If temperatures differ, 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 MluI → complete digest in 15 min at 37 °C (20 µl).
Prolonged incubation/star activity: 3 h at 37 °C shows no nuclease degradation; longer incubations may yield star activity.
Ligation & recutting: After 10× over-digest, >95% ligation at 22 °C; >95% ligated fragments recut by MluI.
Non-specific endonuclease: <10% nicked/linearized plasmid after 4 h at 37 °C.

Methylation Effects

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

Recognition Sites in Common DNAs

DNA	# Sites	DNA	# Sites
λDNA	7	ΦX174	2
pBR322	0	pUC57	0
pUC18/19	0	SV40	0
M13mp18/19	0	Adeno2	5

Activity in Different Buffers

Buffer	Activity
CutOne®	100%
Thermo FastDigest	75%
NEB rCutSmart™	100%
Takara QuickCut™	100%

*Activities are from the functional test conditions described above.

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MluI

Brand: Best Enzymes
Product CAT#: EG15547S
Availability: In Stock

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