DESCRIPTION
Alkaline Phosphatase catalyszes the removal of 5'-phosphate groups from DNA, RNA and ribo- and deoxyribonucleoside triphosphates.
SOURCE
Calf intestinal mucosa.
APPLICATIONS
¡Ü Removing 5' phosphates from DNA or RNA
prior to end labeling 5¡¯ termini with 32P.
¡Ü Removing 5' phosphates from fragments of
DNA to prevent self-ligation.
UNIT DEFINITION
One unit is defined as the amount of enzyme required to catalyze the hydrolysis of 1 ¥ìmol of 4-nitrophenyl- phosphate to 4-nitrophenol in 1 minute at 37¡É.
NOTE
Proteinase K is used to digest CIAP, which must be completely removed if subsequent ligatios are to work efficiently. An alternative method is to activate the CIAP by heating to 65¡É for 1 hour(or 75¡É for 10 minutes) in the presence of 5 mM EDTA(pH 8.0) and then to purify the dephosphorylated DNA by extraction with phenol:chloroform.
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STANDARD UNIT ASSAY CONDITIONS
1 M diethanolamine (pH 9.8), 0.5 mM MgCl2, 10.9 mM 4-nitrophenylphosphate.
CONCENTRATION: 1-5 unit/§¡.
10X REACTION BUFFER
500 mM Tris-HCl (pH 9.3), 10 mM MgCl2, 1 mM ZnCl2, 10 mM spermidine.
STORAGE CONDITIONS
10 mM Tris-HCl (pH £Ü8.0), 1 mM MgCl2, 0.1 mM ZnCl2, 50 mM KCl and 50% glycerol. Store at -20¡É.¡¡
REFERENCE
1. Sambrook, J. and Russell, David W. (2001) Molecular Cloning: A Laboratory Manual, 3rd ed., Cold Spring Harbor Laboratory Press.
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