pKF3 Cloning Test
Quality control of restriction enzyme
The pKF3 Cloning Test is a quality control assay conducted on Takara Bio restriction enzymes which digest a unique site in the multi-cloning site (MCS) of Enforcement Cloning Vector pKF3 DNA. The MCS of pKF3 DNA also encodes the rpsL gene cassette. When rpsL is expressed, it confers a streptomycin (Sm)-sensitive phenotype on the host, which is otherwise Sm resistant. When the restriction enzyme is contaminated with trace amounts of nuclease such as exonuclease, deletion of the rpsL gene cassette occurs, and the host remains resistant to Sm. By taking advantage of this property, even small amounts of nuclease contamination of a restriction enzyme production lot can be detected.
Restriction enzymes adopting pKF3 cloning test
|Acc III||Aor13 HI||Aor51 HI||Ava II||Bal I|
|BamH I||Ban II||Bgl II||BspT 104 I||BstP I|
|Bst1107 I||Cla I||EcoO 65 I||EcoR I||EcoT14 I|
|Eco52 I||Fba I||Hind III||Hpa I||Kpn I|
|Mlu I||Nco I||Nde I||Not I||Nsb I|
|PshB I||Pst I||Pvu I||Pvu II||Sac I|
|Sac II||Sal I||Sma I||Sph I||Sse8387 I|
|Stu I||VpaK 11 BI||Xba I||Xho I|
A slight amount of nuclease such as exonuclease, deletion
occurs in rpsL gene, and host remains to be Sm resistant. By
taking advantage of this property, a small amount of nuclease
contaminating the restriction enzyme can be detected.
The principle of pKF3 cloning test
pKF3 Cloning Test Protocol
- pKF3 DNA is digested by 10-fold excess amount of restriction enzyme which has the unique site in multi-cloning site.
- Following inactivation treatment, part of digested DNA is ligated at 16°C for 30 minutes using DNA Ligation Kit Ver. 1 (Cat.# 6021).
- TH2 Competent Cells are transformed with part of this reaction solution, and cultured over two nights at 37°C on two kinds of plates, i.e., LB-Cm-Sm plate (containing chloramphenicol and streptomycin), and LB-Cm plate (containing chloramphenicol).
When exonuclease, etc., are scarcely contained in the restriction enzyme, colonies are not formed on LB-Cm-Sm plate as the transformant obtained is enforced into streptomycin sensitive. When restriction enzyme is contaminated with exonuclease etc., colonies are formed on LB-Cm-Sm plate as rpsL gene-deficient strain is obtained. The presence or absence of a very minute amount of exonuclease can be judged depending on the presence or absence of colonies appearing on LB-Cm-Sm plate.
Takara has confirmed that the ratio of rpsL gene-deficient transformant (number of colonies appearing on LB-Cm-Sm plate) against transformant (number of colonies appearing on LB-Cm plate) is less than 2%.
Composition of culture
|Yeast Extract||5 mg/ml|
|Yeast Extract||5 mg/ml|