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You are here:Home » Molecular Biology » MB-Enzymes, Restriction » Alw21I (HgiAI)

Alw21I (HgiAI)

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Specifications

5' - G W G C W^C - 3'
3' - C^W C G W G - 5
Catalog #A1372-05
Source Acinetobacter lwoffi RFL21
Concentration 10u/ul
FormSupplied 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 Buffer10mM Tris-HCl pH 7.4, 100mM potassium chloride, 1mM DTT, 1mM EDTA, 0.2mg/ml BSA, 50% glycerol.
Unit DefinitionOne 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 WithR1625: 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
Storage and StabilityMay 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.
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) AssayNo 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
Lambda28
PhiX1743
M13mp18/193
pBR3228
pUC18/195
pUC575
pTZ19R/U4
Thermal Inactivation
Alw21I is inactivated by incubation at 65°C for 20 min.
Recommended Protocol for Digestion
Add
Nuclease free water16ul
10X Restriction Enzyme Buffer D2ul
DNA (0.5-1ug/ul)1ul
Alw21I0.5-2ul
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.
Protocol for Digestion of PCR Products Directly After Amplification
Add
PCR Reaction Mixture10ul (~0.1-0.5ug of DNA)
Nuclease free water18ul
10X Restriction Enzyme Buffer D2ul
Alw21I1-2ul
Mix gently, spin down for a few seconds.
Incubate at 37ºC for 1-16 hours.
Methylation Effects on Digestion
Dam, DcmNever overlaps, no effect
CpG, EcoKIMay overlap, no effect
EcoBIMay overlap, effect not determined


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