SacI (Restriction Enzyme)

REF: EG15565S

Recognition & Cleavage

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

Cleavage pattern:

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

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

Isoschizomers: Psp124BI, SstI, Ecl136II, EcoICRI, Eco53kI (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	EG15565S
SacI (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
Typical duration: 15 min (plasmid); 15–30 min (PCR); 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× CutOne®, ≤1 µg DNA, 1 µl SacI.
30 µl (PCR product): 16 µl ddH₂O, 3 µl 10× CutOne®, ~0.2 µg DNA, 1 µl SacI.
If PCR is unpurified, reduce buffer to 2 µl.
50 µl (genomic DNA): 30 µl ddH₂O, 5 µl 10× CutOne®, 10 µl DNA (5 µg), 5 µl SacI.
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

Use 1 µl of each enzyme for double/multiple digests; total enzyme volume ≤10% of reaction.
If enzymes require different temperatures, start at the lower temperature then shift higher.
Increase incubation time if total volume >20 µl.

Quality Control

Functional test: 1 µg λDNA (HindIII digest) + 1 µl SacI → complete digest in 15 min at 37 °C (20 µl).
Prolonged incubation/star activity: 3 h at 37 °C shows no nuclease degradation; longer may result in star activity.
Ligation & recutting: >95% ligation at 22 °C (T4 DNA ligase); >95% of ligated fragments recut by SacI.
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	2	ΦX174	0
pBR322	0	pUC57	1
pUC18/19	1	SV40	0
M13mp18/19	1	Adeno2	16

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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SacI

Brand: Best Enzymes
Product CAT#: EG15565S
Availability: In Stock

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