BsmBI (Type IIS Restriction Enzyme)

REF: EG22507V

Recognition & Cleavage

Recognition site: 5'-CGTCTC-3' (non-palindromic)

Cleavage pattern:

5' ... C G T C T C (N_¹)^▼ ... 3'
3' ... G C A G A G (N_⁵)_▲ ... 5'

Type: IIS (cuts outside the site) · Common use: Golden Gate assembly

Isoschizomer: Esp3I (methylation sensitivities may differ)

Specs

Optimal temperature: 55 °C
Activity at 37 °C: ~50%
Heat inactivation: 80 °C · 20 min
Storage: −20 °C

Pack Components

Component	EG22507-V	EG22507-S
BsmBI (10 U/µl)	20 µl	100 µl
10× Cut Buffer C	1 ml	1 ml

Recommended Reaction

Buffer: 1× Cut Buffer C (contains recombinant albumin for enzyme stability)
Incubation: 55 °C
Typical load: 5–10 U per µg plasmid DNA; 10–20 U per µg genomic DNA; 1 h water bath
Overnight: Use ~1 U to reduce star activity risk

Definition of 1 U: amount required to completely digest 1 µg λDNA in 50 µl at 50 °C for 1 h.

Quick Protocol (DNA Digestion)

On ice, assemble 50 µl: ddH₂O up to 50 µl, 5 µl 10× Cut Buffer C, 1 µg DNA, 1 µl BsmBI (10 U/µl).
Mix gently, quick-spin.
Incubate at 55 °C for 15–60 min (see enzyme load above).
(Optional) Inactivate at 80 °C for 20 min or clean up (spin column or phenol/chloroform).

Notes

Keep enzyme volume ≤10% of total (glycerol can promote star activity).
Avoid inhibitors in DNA (phenol, chloroform, ethanol, EDTA, high salt, detergents).
Smaller total volumes amplify inhibition from salts/EDTA/glycerol—scale accordingly.

Quality Control

Prolonged incubation/star activity: 10 U with 1 µg λDNA, 3 h at optimal temp → no nonspecific degradation detected (longer incubations may show star activity).
Ligation & recutting: >95% ligation at 22 °C with T4 DNA ligase; >95% of ligated fragments recut by BsmBI.
DNase test: 10 U with 15 ng dsDNA, 16 h at 37 °C → no detectable degradation.

Methylation Effects

Methylase	Effect
Dam	No effect
Dcm	No effect
CpG	Some cleavage blocked
EcoKI	No effect
EcoBI	Cleavage impaired

Recognition Sites in Common DNAs

DNA	# Sites	DNA	# Sites
λDNA	14	ΦX174	0
pBR322	1	pUC57	2
pUC18/19	2	SV40	0
M13mp18/19	1	Adeno2	21

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BsmBI

Brand: Best Enzymes
Product CAT#: EG22507V
Availability: In Stock

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