PacI (Restriction Enzyme)

REF: EG15603S

Recognition & Cleavage

Recognition site: 5′-TTAATTAA-3′ (8-bp palindromic)

Sequence context:

5′ ... T T A A T^▼T A A ... 3′
3′ ... A A T_▲T A A T T ... 5′

Type: II restriction endonuclease · Common use: cloning, verification, rare-cutter mapping. :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	EG15603S
PacI (10 U/µl)	25 µl (250 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
Guideline: Use the fast-digestion setup below; scale volumes as needed.

Color Buffer contains 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 PacI.
30 µl (PCR product): 16 µl ddH₂O, 3 µl 10× buffer, ~0.2 µg DNA, 1 µl PacI.
If PCR is unpurified, use 2 µl 10× buffer due to residual metal ions.
50 µl (genomic DNA): 30 µl ddH₂O, 5 µl 10× buffer, 10 µl DNA (5 µg), 5 µl PacI.
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; load directly if Color Buffer was used. :contentReference[oaicite:4]{index=4}

Double / Multiple Digests

Use ~1 µl of each enzyme; keep total enzyme ≤10% of reaction volume.
If temperatures differ, start at the lower temperature, then add the second enzyme and shift higher.
Increase incubation if total volume >20 µl. :contentReference[oaicite:5]{index=5}

Quality Control

Functional test: 1 µg pPacI DNA + 1 µl PacI 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 result in star activity.
Ligation & recutting: After 10× over-digest, >95% ligation at 22 °C; >95% ligated fragments recut by PacI.
Non-specific endonuclease: <10% nicked/linearized plasmid after 4 h at 37 °C. :contentReference[oaicite:6]{index=6}

Methylation Effects

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

Methylation data. :contentReference[oaicite:7]{index=7}

Recognition Sites in Common DNAs

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

Site counts. :contentReference[oaicite:8]{index=8}

Activity in Different Buffers

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

Activities per the functional test conditions. :contentReference[oaicite:9]{index=9}

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PacI

Brand: Best Enzymes
Product CAT#: EG15603S
Availability: In Stock

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