5' - G W G C W^C - 3' 3' - C^W C G W G - 5’
Source
Acinetobacter lwoffi RFL21
Form
Supplied as a liquid in 10mM Tris-HCl pH 7.5, 300mM sodium chloride, 1mM DTT, 0.1mM EDTA, 0.2mg/ml BSA, 50% glycerol.
Dilution Buffer
10mM Tris-HCl pH 7.4, 100mM potassium chloride, 1mM DTT, 1mM EDTA, 0.2mg/ml BSA, 50% glycerol.
Unit Definition
One unit is defined as the amount of Alw21I required to digest 1ug of lambda DNA in 1 hour at 37°C in 50ul of assay buffer.
Supplied With
R1625: Restriction Enzyme Buffer A, 10X: Supplied as a liquid in 33mM Tris-acetate, 10mM magnesium acetate, 66mM potassium acetate, 0.1mg/ml BSA pH 7.9 at 37°C when diluted to 1X. Note: 1X conc. yields 20-50% enyzme activity. 2X conc. yields 50-100% enzyme activity.
R1625-03: Restriction Enzyme Buffer D, 10X: Supplied as a liquid in 50mM Tris-HCl, pH 7.5, 10mM MgCl2, 100mM sodium chloride, 0.1mg/ml BSA when diluted to 1X. Incubate at 37°C. Note: This buffer yields 100% enzyme activity
Overdigestion Assay
No detectable change in the specific fragmentation pattern is observed after 160-fold overdigestion (10u/ug lambda DNA x 16 hours) with Alw21I.
Ligation/Recutting Assay
After 50-fold overdigestion (3u/ug DNA x 17 hours) with Alw21I, more than 95% of the DNA fragments can be ligated at a 5'-termini concentration of 0.27uM. More than 95% of these sites can be recut.
Labeled Oligonucleotide (LO) Assay
No detectable degradation of a single-stranded and double-stranded labeled oligonucleotide was observed after incubation with 10 units of Alw21I for 4 hours.
Stability During Prolonged Incubation
A minimum of 0.1 units of Alw21I is required for complete digestion of 1ug of lambda DNA in 16 hours at 37°C.
Compatible Ends
GTGCA^C - BseSI, SduI, Mph1103I, PstI, SdaI GAGCT^C - Eco24I, SacI, SduI GAGCA^C - SduI
Number of Recognition Sites in DNA
Lambda: 28 PhiX174: 3 M13mp18/19: 3 pBR322: 8 pUC18/19: 5 pUC57: 5 pTZ19R/U: 4
Thermal Inactivation
Alw21I is inactivated by incubation at 65°C for 20 min.
Add
Nuclease free water: 16ul 10X Restriction Enzyme Buffer D: 2ul DNA (0.5-1ug/ul): 1ul Alw21I: 0.5-2ul Mix gently, spin down for a few seconds. Incubate at 37ºC for 1-16 hours. This digestion reaction may be scaled either up or down.
Add
PCR Reaction Mixture: 10ul (~0.1-0.5ug of DNA) Nuclease free water: 18ul 10X Restriction Enzyme Buffer D: 2ul Alw21I: 1-2ul Mix gently, spin down for a few seconds. Incubate at 37ºC for 1-16 hours.
Methylation Effects on Digestion
Dam, Dcm: Never overlaps, no effect CpG, EcoKI: May overlap, no effect EcoBI: May overlap, effect not determined
Storage and Stability
May be stored at 4°C for short-term only. Aliquot to avoid repeated freezing and thawing. Store at -20°C. Aliquots are stable for 6 months at -20°C. For maximum recovery of product, centrifuge the original vial after thawing and prior to removing the cap.
Source
Acinetobacter lwoffi RFL21
Important Note
This product as supplied is intended for research use only, not for use in human, therapeutic or diagnostic applications without the expressed written authorization of United States Biological.
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