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You are here:Home » Molecular Biology » MB-Enzymes, Restriction » Alw44I (ApaLI)

Alw44I (ApaLI)

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Specifications

5'-G^T G C A C-3'
3'-C A C G T^G-5'
Catalog #A1372-15
Unit DefinitionOne unit is defined as the amount of Alw44l required to digest 1ug of lambda DNA-Smal fragments in 1 hour at 37ºC in 50ul of assay buffer.
Diluent Buffer10mM Tris-HCl, pH 7.4, 100mM KCl, 1mM EDTA, 1mM DTT, 0.2mg/ml BSA, 50% glycerol.
Storage BufferSupplied as a liquid in 10mM Tris-HCl pH 7.5, 50mM KCl, 1mM DTT, 0.1mM EDTA, 0.2mg/ml BSA, 50% glycerol.
Supplied withR1625-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 37°C.
Methylation Effects on Digestion
DamNever overlaps - no effect.
DcmNever overlaps - no effect.
CpGCompletely overlaps - blocked
EcoKlNever overlap - no effect
EcoBlNever overlaps - no effect.
Number of Recognition Sites in DNA
Lambda4
PhiX1741
pBR3223
pUC573
pUC18/193
pTZ19R/U2
M13/mp18/191
Compatible Ends
Bfml
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 37ºC.
Protocol for Digestion
Add
Nuclease free water16ul
R16252ul
DNA (0.5-1ug/ml)1ul
A1372-150.5-2ul
Mix gently and spin down for a few seconds. Incubate at 37ºC for 1-16 hours.
Protocol for Digestion Directly after Amplification
Add
PCR Reaction Mixture10ul (~0.1-0.5ug of DNA)
Nuclease free water18ul
R16252ul
A1372-151-2ul
Mix gently and spin down for a few seconds.
Incubate at 37ºC for 1-16 hours.
Ligation/Recutting AssayAfter 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) AssayNo detectable degradation of a single-stranded and double-stranded labeled oligonucleotide occurred during incubation with 10 units of Alw44l for 4 hours.
Thermal InactivationEnzyme is inactivated by incubation at 65°C for 20 minutes.


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