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You are here:Home » Molecular Biology » MB-Enzymes, Restriction » BseLI (BslI)

BseLI (BslI)

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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
SourceBacillus stearothermophilus LK 3551
Supplied WithR1625: Restriction Enzyme Buffer A, 10X
Concentration 10u/ul
Unit DefinitionOne 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 BufferFor 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 BufferFor 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 StabilityMay 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
DamNever overlaps - no effect
DcmMay overlap - cleavage impaired
CpGMay overlap - cleavage impaired
EcoKlNever overlaps - no effect
EcoBlNever 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
Lambda176
M13mp18/1917
pBR32220
pUC18/19, pUC576
pTZ19R/U7
X17419
NoteBseLI cleavage is impaired by overlapping dcm methylation. To avoid dcm methylation, use a dam, dcm strain such as GM2163.
Quality Control
Overdigestion AssayNo detectable change in the specific fragmentation pattern is observed after 160-fold overdigestion (10u/ug lambda DNA x 16 hours) with BseLI.
Ligation/Recutting AssayAfter 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.


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