Technical Data
A1372-10
Alw26I (BsmAI)
1000U
Molecular Biology Storage: -20CShipping: Blue Ice
5'-G T C T C (N)1^-3'
3'-C A G A G (N)5^-5'

Source:
Acinetobacter lwoffi RFL26

Concentration:
10u/ul

Unit Definition:
One unit is defined as the amount of Alw26I required to digest 1ug of lambda DNA in 1 hour at 37C in 50ul of assay buffer.

Incubation Temperature:
37C

Diluent Buffer:
10mM Tris-HCl, pH 7.4, 100mM KCl, 1mM EDTA, 1mM DTT, 0.2mg/ml BSA and 50% glycerol.

Storage Buffer:
Supplied as a liquid in 10mM Tris-HCl, pH 7.4, 100mM KCl, 1mM DTT, 1mM EDTA, 0.2mg/ml BSA and 50% glycerol.


Restriction Enzyme Buffer A, 10X:
R1625: Supplied as a liquid in 33mM Tris-acetate, pH 7.9, 10mM magnesium acetate, 66mM potassium acetate and 0.1mg/ml BSA. Incubate at 37C. Supplied to simplify buffer selection for double digests.

Storage and Stability:
May be stored at 4C for short-term only. For long-term storage, aliquot and store at -20C. Aliquots are stable for at least 6 months at -20C. 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.
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 Alw26I.

Ligation/Recutting Assay:
After 50-fold overdigestion (3u/ug DNA x 17 hours) with Alw26I more than 95% of the DNA fragments can be ligated at a 5'-termini concentration of 0.1uM. 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 Alw26I for 4 hours.

Methylation Effects:
Alw26I does not cut GAGAm5C. Overlapping CpG methylation may influence DNA cleavage.

Stability during Prolonged Incubation:
A minimum of 0.2units of Alw26I is required for complete digestion of 1ug of lambda DNA in 16 hours at 37C.

Thermal Inactivation:
Alw26I is inactivated by incubation at 65C for 20min.

Number of Recognition Sites in DNA:
Lambda: 37
PhiX174: 4
M13mp18/19: 5
pBR322: 3
pUC18/19: 4
pUC57: 4
pTZ19R/U: 2

Protocol for Digestion:
Add:
Nuclease free water: 16ul
R1625: 2ul
DNA (0.5-1ug/ml): 1ul
A1372-10: 0.5-2ul
Mix gently and spin down for a few seconds. Incubate at 37C for 1-16 hours.

Protocol for Digestion Directly after Amplification:
Add:
PCR Reaction Mixture: 10ul (~0.1-0.5ug of DNA)
Nuclease free water: 18ul
R1625: 2ul
A1372-10: 1-2ul
Mix gently and spin down for a few seconds.
Incubate at 37C for 1-16 hours.
Intended for research use only. Not for use in human, therapeutic, or diagnostic applications.