Product info: Thermolabile Alkaline Phosphatase


Name
Thermolabile Alkaline Phosphatase
Cat. #E365E366
Package, u.a.2001000
Concentration, u.a./ml50005000

SourceIsolated from E.coli strain that carries the cloned Alkaline Phosphatase gene from Alteromonas undina P2
DescriptionThermolabile Alkaline Phosphatase (TAP) catalyzes the dephosphorylation of 5' and 3' ends of DNA and RNA. Also, TAP hydrolyzes ribo- and deoxyribonucleoside triphosphates.
Applications:
-removing 5` and 3` phosphoryl groups from nucleic acids;
-preparing templates for 5` end labeling;
-preventing DNA fragments from self-ligating.
Unit definitionOne unit is defined as the amount of enzyme that hydrolyzes 1 μmol of 4-nitrophenylphosphate (PNPP) in a total reaction volyme of 0.5 ml in15 min at 16°C . Enzyme activity is assayed in the following mixture: 1X SE-buffer W, 10 mM 4-nitrophenylphosphate.
Reaction bufferSE-buffer W,(10 mM Tris-HCl (pH 8.5 at 25°C); 10 mM MgCl2; 100 mM NaCl; 1 mM DTT.)
Optimal temperature
16oC
Storage conditions20 mM Tris-HCl (pH 7.6), 0.1 mM ZnCl2, 50% glycerol. Store at -20°C
Inactivation 20 minutes under
65oC
NotesFunctional assay: As a result of treatment 1μg of pUC19/Hind III DNA containing 1X SE-buffer W and 1μl of the enzyme in a total reaction volume of 20 μl in 30 min at 16°C , with following ligation with T4 DNA Ligase and transformation of E.coli cells, >10-times decreasing of clones amount was achieved compared to use of untreated pUC19/Hind III DNA.
Protocol for dephosphorilation of 5'-ends of DNA digested with Restrction Endonuclease (RE) using TAP:
20 μl of the reaction volume:
10X SE-buffer for RE or Buffer W -2 μl
DNA - 0,5-1 μg
Nuclease-free water -to 20 μl
Add 1 μl of RE, mix by pipetting.
Incubate for 0,5-1 hour at optimal temperature.
Chill the reaction mixture on ice for 1-2 min!!! Add 1 μl (5 units) of TAP (E365/E366), mix by pipetting. Incubate for 0,5-1 hour at 16°C (Incubation at 25°C results in 75-100% activity). Heat the reaction mixture for 20 min at 65°C. If necessary, make precipitation of DNA with ethanol or purify it by gel-filtration or spin-column.
Quality controlThe enzyme is purified free of contaminating endonucleases and exonucleases, ribonuclease activities.
MSDS:Download MSDS as PDF