CIP Treatment
1. set up the following reaction:
| CIP Rxn | |
| H 2 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 H 2 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
|
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 |
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