西亚试剂：：CIP Treatment

1. incubate for 60 min at 37°C
2. add 1.14 ml 0.2 M trinitriloacetic acid/NaOH pH 8.0
3. heat inactivate for 10 min at 70°C
4. phenolize 3x
5. do an EtOH precipitation and take up pellet in 10 ml H₂O (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. states that one should use 1.0 unit/pmol DNA ends. 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