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5'-G^T G C A C-3'
3'-C A C G T^G-5'
One 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.
10mM Tris-HCl, pH 7.4, 100mM KCl, 1mM EDTA, 1mM DTT, 0.2mg/ml BSA, 50% glycerol.
Supplied as a liquid in 10mM Tris-HCl pH 7.5, 50mM KCl, 1mM DTT, 0.1mM EDTA, 0.2mg/ml BSA, 50% glycerol.
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 37°C.
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:
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.
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:
Nuclease free water: 16ul
DNA (0.5-1ug/ml): 1ul
Mix gently and spin down for a few seconds. Incubate at 37ºC for 1-16 hours.
Protocol for Digestion Directly after Amplification:
PCR Reaction Mixture: 10ul (~0.1-0.5ug of DNA)
Nuclease free water: 18ul
Mix gently and spin down for a few seconds.
Incubate at 37ºC 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 65°C for 20 minutes.