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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.

+ Phosphatase →

Usage

  1. To supress self-ligation of cloning vector.
  2. To prereact for labelling 5'-terminus with γ-32P.

Procedure

CIAP (Calf Intestine Alkaine Phosphatase) and BAP(Bovine alkaline phosphatase) are commercially available.

BAP and CIAP

BAP

  1. Disolve DNA in 17μL of ddH2O
  2. Add 2μL of 10x buffer
  3. Add 1μL of BAP
  4. Incubate at 60-65℃ for 1hr.

CIAP

  1. Disolve DNA in 17μL of ddH2O
  2. Add 2μL of 10x buffer
  3. Add 1μL of CIAP
  4. Incubate at 37℃ for 1 hr.
    • (or 37℃/15min + 50℃/15min)

Caution

  1. 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/CHCl3 extraction method. Sometimes heat-inactivation is not enough to remove the phosphatase activity in the solution.

Hints

  1. Kinase with ADP also can dephosphorylate.