CIP Treatment

1. set up the following reaction:
   
   

   CIP Rxn
   H2 O	7.8 ml
   10x cip rxn buffer	2.0 ml
   DNA (e.g; 3 kb vector; 0.2 mg/ml; 2 mg total)	10.0 ml
   (1 u/ml) CIP	0.2 ml
   total	20.0 ml

   
    
2. incubate for 60 min at 37°C
3. add 1.14 ml 0.2 M trinitriloacetic acid/NaOH pH 8.0
4. heat inactivate for 10 min at 70°C
5. phenolize 3x
6. do an EtOH precipitation and take up pellet in 10 ml H₂ O (0.2 mg/ml)

Solutions:

10x CIP Buffer: 0.5 M Tris-HCl pH 8.5, 1 mM EDTA, pH 8.5 (supplied with enzyme) nitrilotriacetic acid (also known as trinitriloacetic acid): adjust to pH 8.0 with 5 N NaOH, an excellent chelator
CIP Buffer (10x)   1 M Tris-HCl pH 8.5 500.0 ml 0.5 M EDTA pH 8.0 2.0 ml H2O 498.0 ml Total 1.0 ml

Remarks:

In principle, one needs 1u CIP/100 pmol protruding 5' ends and 1u CIP/2pmol for blunt or recessed termini (Sambrook). 1 mg of a 3 kb vector corresponds to 0.5 pmol 5' ends. Different suppliers give different advice. NEB states that one should use 1.0 unit/pmol DNA ends. Amersham Pharmacia Biotech suggests using 0.1 units for 1-20 mg of DNA.

Materials:

Reagent/Tool	Supplier	Cat.-#
CIP (or CIAP)	BMG	713 023
nitrilotriacetic acid (disodium salt)	Sigma	N-0128