USE: Decrease of vector background in cloning strategies:

Alkaline phosphatase catalyses the removal of 5'-phosphate groups from DNA, RNA and ribo- and deoxyribonucleoside triphosphates.

Since CIP-treated fragments lack the 5'-phosphoryl termini required by ligases, they cannot self-ligate.

Applications:

Removing 5' phosphates from DNA, RNA. rNTPs and dNTPs

Preventing recircularisation of cloning vectors

Reaction buffer:

CIP works in the following NEB buffers: 2, 3, 4, EcoRI, BamHI, SalI

(1x CIP-buffer: 100 mM NaCl, 50 mM Tris-Hcl, 10 mM MgCl2, 1 mM DTT, pH 7.9)

Dephosphorylating with CIP:

2. Easy protocol: Add 0.5 µl CIP in 50 µl final volume and incubate 20 min at 37°C (for detailed protocol, see NEB-catalogue)

3. Gel purify DNA or Phenol chloroform extract

4. Optional in case of Gel purification:

Recover DNA by ethanol precipitation

Use the Promega enzyme (CIAP) and protocol

This protocol involves two sequential incubations at 37°C for 30 min with low amounts of CIP (see below) in 50 µl volume, followed by phenol/chloroform extraction and ethanol-precipitation.

see in Mol.Biol. folder or on Promega website

Note: 1 pmole of ends of linear DNA is equivalent to 1.6 µg of a 4.4 kb vector. In general use roughly 0.5 U CIAP up to 10 pmoles of ends. (Promega even suggests as little as 0.01 U per pmole.)

Example: 6 µg of an 8.8 kb linearised vector will correspond to ca. 2pmole of ends (8.8/4.4 = 2x bigger -> 1pmole = 2x1.6µg=3.2µg)