Invitrogen™ CIAP (Calf Intestinal Alkaline Phosphatase), 1 U/μL

Catalog No.	Quantity
18-009-027	1000 U

Catalog No. 18-009-027 Supplier Invitrogen™ Supplier No. 18009027

Description

Phosphomonoesterase that removes 3' and 5' phosphates from DNA and RNA

• Concentration: 20 units/μL
• Source: Purified from calf intestinal mucosa
• Performance and Quality Testing: Endodeoxyribonuclease, 3' exodeoxyribonuclease, and ribonuclease assays; dephosphorylation efficiency measured in a transformation assay
• Unit Definition: One unit hydrolyzes 1μmol of 4-nitrophenyl phosphate in 1 min. at 37°C
• Unit Reaction Conditions: 1M diethanolamine buffer, 10mM 4-nitrophenyl phosphate, 0.25mM MgCl₂ (pH 9.8) in 900μL for 10 min. at 37°C

Dephosphorylation of 5'-phosphorylated termini of vector DNA to prevent self-ligation; Dephosphorylation of 5' termini of nucleic acids prior to forward reaction with kinase; Cloning; Restriction Enzyme Cloning

Specifications

• Concentration: 1 U/μL
• Content And Storage: Calf Intestinal Alkaline Phosphatase (1 unit/μL) is supplied with a vial of 10X dephosphorylation buffer [500 mM Tris-HCl (pH 8.5), 1 mM EDTA], vial of dilution buffer [50% glycerol, 25 mM Tris-HCl (pH 7.6), 1 mM MgCl₂ , and 0.1 mM ZnCl₂ ]. Store Calf Intestinal Alkaline Phosphatase at -20°C.
• Shipping Condition: Approved for shipment on Wet or Dry Ice
• Enzyme: CIP
• Compatible Buffer: Diethanolamine Buffer
• Quantity: 1000 U
• Product Type: Alkaline Phosphatase

Frequently Asked Questions (FAQs)

What other enzymes besides ligase may I need to perform restriction cloning?

Dephosphorylating the vector to decrease background can be achieved with:
Heat Inactivated Alkaline Phosphatase, or Calf Intestinal Alkalline Phosphatase (available as 1 unit/µL or 20 units/µL)

If you need to create blunt phosphorylated DNA ends (“polishing” the ends), you can use:
DNA End Repair Mix or T4 DNA polymerase or Klenow Fragment (large fragment) of E. coli DNA polymerase to generate blunt ends due to their 5' to 3' DNA polymerase activity (filling-in of 5' overhangs) and 3' to 5' exonuclease activity (chewing back of 3' overhangs).

What enzymes can be used to remove 5' phosphate groups, and what enzyme can be used to add the phosphate groups back on?

CIP/CIAP or BAP can dephosphorylate the 5' ends of DNA/RNA. T4 Polynucleotide Kinase can add back phosphate groups.

Why are alkaline phosphatases used in cloning protocols? What is a typical protocol for dephosphorylation of nucleic acids?

Alkaline phosphatases are used to dephosphorylate the 5' ends of DNA. In cloning, it is used to prevent self-ligation of vector DNA. Standard ligation of DNA with ligase requires a 5' phosphate to be present on at least one of the ends being joined. When a DNA insert containing phosphates on both 5' termini is added to a dephosphorylated vector, the insert will be efficiently ligated into the vector, but the vector will not be able to self-ligate. Thus, dephosphorylation of vector lowers the number of background colonies containing vector without insert.

For Research Use Only. Not for use in diagnostic procedures.