Technical Data
Alw44I (ApaLI)
Molecular Biology Storage: -20CShipping: Blue Ice
5'-G^T G C A C-3'
3'-C A C G T^G-5'

Unit Definition:
One unit is defined as the amount of Alw44l required to digest 1ug of lambda DNA-Smal fragments in 1 hour at 37C in 50ul of assay buffer.

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

Storage Buffer:
Supplied as a liquid in 10mM Tris-HCl pH 7.5, 50mM KCl, 1mM DTT, 0.1mM EDTA, 0.2mg/ml BSA, 50% glycerol.

Supplied with:
R1625-Restriction Enzyme Buffer A (double digests): 1X Buffer composition-Supplied as a liquid containing 33mM Tris-acetate pH 7.9, 10mM magnesium acetate, 66mM potassium acetate, 0.1mg/ml BSA. Incubate at 37C.
Methylation Effects on Digestion:
Dam: never overlaps - no effect.
Dcm: never overlaps - no effect.
CpG: completely overlaps - blocked
EcoKl: never overlap - no effect
EcoBl: never overlaps - no effect.

Number of Recognition Sites in DNA:
Lambda: 4
PhiX174: 1
pBR322: 3
pUC57: 3
pUC18/19: 3
pTZ19R/U: 2
M13/mp18/19: 1

Compatible Ends:

Digestion of Agarose-embedded DNA:
A minimum of 5 units of the enzyme is required for complete digestion of 1ug of agarose-embedded lambda DNA in 16 hours.

Overdigestion Assay:
No detectable change in the specific fragmentation pattern is obseAlw44l..
rved after 160-fold overdigestion (10u/ug lambda DNA x 16 hours) with Alw44I

Stability during Prolonged Incubation:
A minimum of 0.1 units of the Alw44I is required for complete digestion of 1ug of DNA in 16 hours at 37C.

Protocol for Digestion:
Nuclease free water: 16ul
R1625: 2ul
DNA (0.5-1ug/ml): 1ul
A1372-15: 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:
PCR Reaction Mixture: 10ul (~0.1-0.5ug of DNA)
Nuclease free water: 18ul
R1625: 2ul
A1372-15: 1-2ul
Mix gently and spin down for a few seconds.
Incubate at 37C for 1-16 hours.

Ligation/Recutting Assay: After 50-fold overdigestion (3u/ug DNA x 17 hours) with Alw44I more than 90% of the DNA fragments can be ligated at a 5'-termini concentration of 0.03uM. More than 95% of these can be recut.

Labeled Oligonucleotide (LO) Assay: No detectable degradation of a single-stranded and double-stranded labeled oligonucleotide occurred during incubation with 10 units of Alw44l for 4 hours.

Thermal Inactivation: Enzyme is inactivated by incubation at 65C for 20 minutes.
Intended for research use only. Not for use in human, therapeutic, or diagnostic applications.