|Product info: Alkaline Phosphatase|
|Cat. #||E327||E328 |
|Package, u.a.||100||500 |
|Concentration, u.a./ml||10000||10000 |
|Source||Calf intestinal mucosa|
|Description||Alkaline Phosphatase, Calf Intestinal (CIP) catalyzes the dephosphorylation of 5` and 3` ends of DNA and RNA. Also, CIP hydrolyses 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 volume of 1 ml in 1 min at 37°C. Enzyme activity quantitation is assayed in the following mixture: 1 M Diethanolamine-HCl (pH 9.8 @ 25°C), 10 mM 4-nitrophenylphosphate, 0.5 mM MgCl2.
|Reaction buffer||SE-buffer O,(50 mM Tris-HCl (pH 7.6 at 25°C); 10 mM MgCl2; 100 mM NaCl; 1 mM DTT.)|
|Storage conditions||10 mM Tris-HCl (pH 8.2 @25°C),50 mM KCl,1 mM MgCl2, 0.1 mM ZnCl2, 50% glycerol. Store at -20°C|
|Notes||Functional assay: As a result of treatment 1 μg of pUC19/HindIII DNA containing 1 x SE-buffer O and 0.5μl of the enzyme in a total reaction volume of 20 μl in 30 min at 37°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/HindIII DNA.|
Protocol for dephosphorilation of 5`-ends of DNA digested with Restriction Endonuclease (RE) using CIP:
20 μl of the reaction volume:
10 x SE-buffer for RE or Buffer O - 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.
Add 0,2-1 μl (2-10 units) of CIP (E327/E328), mix by pipetting.
Incubate for 0,5-1 hour at 37°C.
Purify DNA by gel-filtration, spin-column or phenol/chloroform extraction with following ethanol precipitation.
|Quality control||The enzyme is purified free of contaminating exonuclease, endonuclease, ribonuclease activities.|
|MSDS:||Download MSDS as PDF|