|Molecular Biology||Storage: -20°CShipping: Blue Ice|
5'-G T C T C (N)1^-3'
3'-C A G A G (N)5^-5'
Acinetobacter lwoffi RFL26
One unit is defined as the amount of Alw26I required to digest 1ug of lambda DNA in 1 hour at 37°C in 50ul of assay buffer.
10mM Tris-HCl, pH 7.4, 100mM KCl, 1mM EDTA, 1mM DTT, 0.2mg/ml BSA and 50% glycerol.
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 37°C. Supplied to simplify buffer selection for double digests.
Storage and Stability:
May be stored at 4°C for short-term only. For long-term storage, aliquot and store at -20°C. Aliquots are stable for at least 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.
No detectable change in the specific fragmentation pattern is observed after 160-fold overdigestion (10u/ug lambda DNA x 16 hours) with Alw26I.
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.
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 37°C.
Alw26I is inactivated by incubation at 65°C for 20min.
Number of Recognition Sites in DNA:
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.