|Source||Isolated from E.coli strain that carries the cloned Alkaline Phosphatase gene from Alteromonas undina P2|
|Description||Thermolabile Alkaline Phosphatase (TAP) catalyzes the dephosphorylation of 5' and 3' ends of DNA and RNA. Also, TAP hydrolyzes ribo- and deoxyribonucleoside triphosphates.|
-removing 5` and 3` phosphoryl groups from nucleic acids;
-preparing templates for 5` end labeling;
-preventing DNA fragments from self-ligating.
|Unit definition||One 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 buffer||SE-buffer W,(10 mM Tris-HCl (pH 8.5 at 25°C); 10 mM MgCl2; 100 mM NaCl; 1 mM DTT.)|
|Storage conditions||20 mM Tris-HCl (pH 7.6), 0.1 mM ZnCl2, 50% glycerol. Store at -20°C|
|Inactivation 20 minutes under||65oC|
|Notes||Functional 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
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 control||The enzyme is purified free of contaminating endonucleases and
exonucleases, ribonuclease activities.|
|MSDS:||Download MSDS as PDF|