CIP, Calf Intestinal Alkaline Phosphatase

A high quality enzyme for removing 5´ phosphates from DNA and RNA

Description

Alkaline phosphatase catalyses the removal of 5’ phosphate residues from DNA, RNA and ribo- and deoxyribo­nucleoside triphosphates. CIP is primarily used to remove 5’ phosphates from DNA fragments to prevent self-ligation and to remove 5’ phosphates from DNA and RNA prior to labeling the 5’ end with ³²P. Alkaline phosphatase is purified free of exonucleases, endonucleases and RNases. The quality is confirmed with versatile QC tests which also include transformation assay.

Applications

• Removal of 5´ phosphates from DNA fragments to prevent self-ligation
• Removal of 5´ phosphates from DNA and RNA prior to labeling the 5´end with ³²P

Advantages

• Highly purified product
• High quality: QC tests include transformation assay
• Works with all standard restriction enzyme buffers

Concentration and activity

The concentration of CIP is 10 U/µl and the specific activity is >2000 U/mg. CIP is supplied with optimized 10x CIP Buffer. 1x buffer contains: 10 mM Tris-HCl (pH 7.9 at 25°C), 10 mM MgCl₂, 1 mM DTT and 50 mM NaCl.

Information for users

When dephosphorylating use 0.1 U CIP / 1 pmol DNA ends. CIP can be inactivated by heating (5 mM EDTA,
75°C, 10 min or 65°C, 60 min). Excess amounts of CIP may cause co-precipitation of DNA when inactivating CIP by heat treatment.

Quality control

Unit definition
One unit is defined as the amount of enzyme that hydrolyses 1 µmol of p-nitrophenylphosphate to p-nitrophenol in 1 minute at 37 °C.

Unit assay conditions
1 M diethanolamine-HCl (pH 9.8), 0.5 mM MgCl₂, 10 mM p-nitrophenylphosphate.

16-hour incubation
A 50 µl reaction containing 1 µg of λ DNA and 5 U of enzyme incubated for 16 hours at 37 °C resulted in the same DNA band as a reaction produced without the enzyme.

Exonuclease activity
Incubation of 10 U for 4 hours at 37 °C in 50 µl assay buffer with 1 µg sonicated [³H]-DNA (2 x 10⁵ cpm/µg) released <0.5 % of radioactivity.

Endonuclease activity
Incubation of 10 U with 1 µg φX174 RFI DNA (4 hours, 37 °C, 50 µl) gave <5 % conversion to RFII.

RNase activity
Incubation of 10 U with 1 µg MS2 RNA (4 hours, 37 °C, 50 µl) resulted in the same RNA band as that produced without the enzyme.

Transformation assay
pUC19 DNA was linearized with Hind III restriction endonuclease. The DNA was then treated with 0.01 U Calf Intestinal Alkaline Phosphatase per pmol 5’ends at 37 °C for 60 minutes. After phenol/chloroform extraction and ethanol precipitation the ligation efficiency of the DNA was tested with and without kinase treatment. The ability to ligate an insert to the vector was also tested. After ligation the DNA was transformed into competent cells and plated onto plates containing X-Gal and IPTG.

Storage and stability

Recommended storage temperature -20°C. Stable for one year from the assay date.

Ordering information

CIP	Size	Concentration
F-201S	1 000 U	10 U/µl
F-201L	5 000 U	10 U/µl