SacII (Restriction Enzyme)

REF: EG15605S

Recognition & Cleavage

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

Sequence context:

5′ ... C C G C^▼G G ... 3′
3′ ... G G_▲C G C C ... 5′

Type II restriction endonuclease for routine cloning & mapping. Isoschizomers: Cfr42I, KspI, Sfr303I, SgrBI (methylation sensitivities may differ). :contentReference[oaicite:1]{index=1}

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	EG15605S
SacII (20 U/µl)	50 µl (1000 U)
10× CutOne® Buffer	1 ml
10× CutOne® Color Buffer	1 ml

Specs & components: :contentReference[oaicite:2]{index=2}

Recommended Reaction

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

Color Buffer includes density reagent with red/yellow tracking dyes for direct gel loading. :contentReference[oaicite:3]{index=3}

Quick Protocol (Fast DNA Digestion)

20 µl (plasmid): 15 µl ddH₂O, 2 µl 10× buffer, ≤1 µg DNA, 1 µl SacII.
30 µl (PCR product): 16 µl ddH₂O, 3 µl 10× buffer, ~0.2 µg DNA, 1 µl SacII. If PCR not purified, use 2 µl 10× buffer.
50 µl (genomic DNA): 30 µl ddH₂O, 5 µl 10× buffer, 10 µl DNA (5 µg), 5 µl SacII.
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 different temperatures are needed, start lower then shift higher for the second enzyme.
Increase incubation time if total volume >20 µl.

Protocol details: :contentReference[oaicite:4]{index=4}

Quality Control

Functional test: 1 µg pSacII DNA + 1 µl SacII 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 incubation may cause star activity.
Ligation & recutting: After 10× over-digest, >95% ligation at 22 °C; >95% of ligated fragments recut.
Non-specific endonuclease: <10% nicked/linearized plasmid after 4 h at 37 °C.

QC summary: :contentReference[oaicite:5]{index=5}

Methylation Effects

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

Methylation data: :contentReference[oaicite:6]{index=6}

Recognition Sites in Common DNAs

DNA	# Sites	DNA	# Sites
λDNA	4	ΦX174	1
pBR322	0	pUC57	0
pUC18/19	0	SV40	0
M13mp18/19	0	Adeno2	33

Site counts: :contentReference[oaicite:7]{index=7}

Activity in Different Buffers

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

Buffer activity: :contentReference[oaicite:8]{index=8}

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SacII

Brand: Best Enzymes
Product CAT#: EG15605S
Availability: In Stock

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