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en:phosphatase [2013/06/09 10:10] (current)
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 +<select Language>​
 +bio:​ホスファターゼ|日本語
 +bio:​en:​Phosphatase|English
 +</​select>​
 +
 +======Polynucleotide phosphatase======
 +Polynucleotide phosphatase has an activity of desphosphorylation of polynucleotide. In cloning technique, CIAP (Calf Intestine Alkaine Phosphatase) or BAP(Bovine alkaline phosphatase) are often used because of their unifunctionality.
 +
 +
 +
 +{{bio:​phospho_term5.png|}} + Phosphatase -> {{bio:​hydroxy_term5.png|}}
 +
 +
 +
 +
 +
 +=====Usage=====
 +  -To supress self-ligation of cloning vector.
 +  -To prereact for labelling 5'​-terminus with γ-<​sup>​32</​sup>​P.
 +
 +=====Procedure=====
 +CIAP (Calf Intestine Alkaine Phosphatase) and BAP(Bovine alkaline phosphatase) are commercially available.
 +====BAP and CIAP====
 +
 +**BAP**
 +  -Disolve DNA in 17μL of dd<​chem>​H2O</​chem>​
 +  -Add 2μL of 10x buffer
 +  -Add 1μL of BAP
 +  -Incubate at **60-65℃** for 1hr.
 +  -[[PCI extraction]] x 2
 +  -[[Ethanol precipitation]]
 +
 +
 +**CIAP**
 +  -Disolve DNA in 17μL of dd<​chem>​H2O</​chem>​
 +  -Add 2μL of 10x buffer
 +  -Add 1μL of CIAP
 +  -Incubate at **37℃** for 1 hr.
 +    *(or **37℃**/​15min ​ + **50℃**/​15min)
 +  -[[PCI extraction]]
 +  -[[Ethanol precipitation]]
 +
 +
 +====Caution====
 +  -Remaining of phosphatase at ligation reaction causes fatal supression of the ligation reaction because the reaction requires the phophorylation of termini with [[ATP]]. Remove the phosphatase activity by using Phenol/​CHCl<​sub>​3</​sub>​ extraction method. Sometimes heat-inactivation is not enough to remove the phosphatase activity in the solution.
 +=====Hints=====
 +  -[[Kinase]] with [[ADP]] also can dephosphorylate.
 +
 +
 +{{tag>​phosphatase protocol}}