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Specifications

5'-C C N N N N N^N N G G-3'
3'-G G N N^N N N N N C C-5'
Catalog #	B2908
Source	Bacillus stearothermophilus LK 3–551
Supplied With	R1625: Restriction Enzyme Buffer A, 10X
Concentration	10u/ul
Unit Definition	One unit is defined as the amount of BseLI required to digest 1ug of lambda DNA dcm-in 1 hour at 55°C in 50ul of assay buffer.
Diluent Buffer	For short term storage (3-4 weeks)- dilute with 10mM Tris-HCl pH 7.4, 100mM KCl, 1mM EDTA, 1mM DTT, 0.2mg/ml BSA and 50% glycerol.
Storage Buffer	For long term storage: 10mM Tris-HCl pH 7.4, 100mM KCl, 1mM DTT, 1mM EDTA, 0.2mg/ml BSA and 50% glycerol.
R1625	Restriction Enzyme Buffer A, 10X:
	Suplied as a liquid in 33mM Tris-acetate pH 7.9, 10mM magnesium acetate, 66mM potassium acetate, 0.1mg/ml BSA.
Storage and Stability	May be stored at 4°C for short-term only. For long-term storage, store at -20°C. Aliquots are stable for 6 months at -20°C. For maximum recovery of product, centrifuge the original vial after thawing and prior to removing the cap. Further dilutions can be made in assay buffer.
Enzyme Properties
Methylation Effects
Dam	Never overlaps - no effect
Dcm	May overlap - cleavage impaired
CpG	May overlap - cleavage impaired
EcoKl	Never overlaps - no effect
EcoBl	Never overlaps - no effect
Stability during Prolonged Incubation
A minimum of 0.1units of BseLI is required for complete digestion of 1ug of lambda DNA in 16 hours at 55°C.
Thermal Inactivation
BseLI is inactivated by incubation at 80°C for 20min.
Inactivation Procedure
To prepare the digested DNA. for electrophoresis
1. Stop the digestation rection by adding 0.5M EDTA, pH8, to achieve a 20mM final concentration. Mix thoroughly, add an electrophoresis loading dye and load onto gel.
To prepare DNA suitable for further enzymatic reactions
1. Extract with phenol/ chloroform, precipitate with ethanol or isopropanol, wash the pellet with 75% cold ethanol and let air dry.
2. Dissolve DNA in either nuclease free water, TE buffer or a buffer suitable for further applications. Check the DNA concentration in the solution.
Number of Recognition Sites in DNA
Lambda	176
M13mp18/19	17
pBR322	20
pUC18/19, pUC57	6
pTZ19R/U	7
X174	19
Note	BseLI cleavage is impaired by overlapping dcm methylation. To avoid dcm methylation, use a dam, dcm strain such as GM2163.
Quality Control
Overdigestion Assay	No detectable change in the specific fragmentation pattern is observed after 160-fold overdigestion (10u/ug lambda DNA x 16 hours) with BseLI.
Ligation/Recutting Assay	After 50-fold overdigestion (3u/ug DNA x 17 hours) with BseLI, more than 95% of the DNA fragments can be ligated at a 5'-termini concentration of 1.6uM. More than 95% of these can be recut.
Labeled Oligonucleotide (LO) Assay
No detectable degradation of a single-stranded and double-stranded labeled oligonucleotide was observed after incubation with 10units of restriction endonuclease for 4 hours.