Calf Intestinal (CIP) Alkaline Phosphatase for Nucleic Acid Dephosphorylation

(CIP)
From Calf Intestine

(EC 3.1.3.1)
Product No. P4978
Store at –20 °C

Product Description
Product Summary
Storage buffer
Procedure: Dephosphorylation of DNA
Heat Inactivation
Materials
References

Product Description

CIP is commonly used to remove 5’-phosphate groups from DNA, RNA and both ribo and deoxy-ribonucleoside triphosphates. Removal of 5’-phosphates is very useful in preventing self-ligation of cleaved DNA vectors. This property greatly reduces background (plasmids without insert) in cloning procedures.

Product Summary

Activity: approx. 10,000 units/mL (see label for lot specific activity)
DNase: None detected
Nickase: None detected
RNase: None detected
Unit Definition: One unit will hydrolyze 1.0 µmole of 4-nitrophenyl phosphate per minute at pH 9.8 at 37 °C (diethanolamine).

10X CIP Buffer (C 3225)
1 M NaCl
0.5 M Tris-HCl pH 7.9 at 25 °C
0.1 M MgCl2
0.01 M dithiothreitol

Storage buffer

10 mM Tris-HCl, pH 8.2
50 mM KCl
1 mM MgCl2
0.1 mM ZnCl2
50 % glycerol

Procedure: Dephosphorylation of DNA

  1. Dissolve DNA in 1X CIP Buffer (0.5µg DNA/10 µL).
  2. For 5’ overhang DNA add 0.1 units/pmol CIP; for 3’ overhang or blunt end DNA add 1 unit/pmol.
  3. Incubate 60 minutes at 37 °C.
  4. Extract with phenol/chloroform (P3803 or P2069) or gel purify the DNA.*
  5. Recover the DNA by alcohol precipitation.
    *Note: Phenol extraction or gel purification makes heat inactivation unnecessary.

Heat Inactivation

Greater than 95% of the activity can be inactivated by heating to 75 °C for 10 minutes in the presence of 5 mM EDTA.

Materials
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References

1.
Moessner E, Boll M, Pfleiderer G. 1980. Purification of Human and Bovine Alkaline Phosphatases by Affinity Chromatography. Hoppe-Seyler´s Zeitschrift für physiologische Chemie. 361(1):543-550. http://dx.doi.org/10.1515/bchm2.1980.361.1.543
2.
Sambrook, et al. J. 1989. Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory.