Restriction enzyme quality control

In order to maintain high-quality standards for our restriction enzymes, we perform a pKF3 cloning test by digesting a unique site in the multiple cloning site (MCS) of the 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 will be detected.

The table below lists the restriction enzymes that we quality test using the pKF3 cloning test:

pKF3 Cloning Test Principle

pKF3 Cloning Test Protocol

1. pKF3 DNA is digested by a 10-fold excess amount of restriction enzyme that has a unique site in the MCS.
2. Following inactivation treatment, a portion of the digested DNA is ligated at 16°C for 30 minutes using DNA Ligation Kit Ver. 1.
3. TH2 Competent Cells are transformed with a portion of the reaction solution and cultured over two nights at 37°C on two kinds of plates, such as LB-Cm-Sm plates (containing chloramphenicol and streptomycin), and LB-Cm plates (containing chloramphenicol).
   
   In the absence of exonuclease contamination, colonies will not appear on the LB-Cm-Sm plate, as the transformant obtained is streptomycin sensitive. When contamination is present, colonies on LB-Cm-Sm plate will be rpsL gene-deficient bacteria. The presence or absence of a minute amount of exonuclease can be determined by the presence or absence of colonies on the LB-Cm-Sm plate.
   
   In order to pass our quality assurance testing, the ratio of rpsL gene-deficient transformant (number of colonies appearing on the LB-Cm-Sm plate) vs. transformant (number of colonies appearing on the LB-Cm plate) must be less than 2%.

Culture Composition 

LB-Cm-Sm plate
Yeast Extract: 5 mg/ml
Pepton: 10 mg/ml
NaCl: 5 mg/ml
Chloramphenicol: 30 µg/ml
Streptomycin: 50 µg/ml
Agar: 1.5%

LB-Cm plate
Yeast Extract: 5 mg/ml
Pepton: 10 mg/ml
NaCl: 5 mg/ml
Chloramphenicol: 30 µg/ml
Agar: 1.5%

We perform a ligation and recutting assay in order to assess and maintain the functional purity of our enzymes. Any contamination by ligase inhibitors, phosphatases, or exonucleases will be detected by this assay: First, the substrate DNA is overdigested by applying a 2- to 50-fold excess amount of restriction enzyme. The digested DNA is then recovered and diluted in T4 DNA Ligase Buffer [66 mM Tris-HCl (pH7.6), 6.6 mM MgCl₂, 10 mM DTT, 0.4 mM ATP] at a 5'-termini concentration of 0.1–1.0 µM. After a sufficient amount of T4 DNA ligase has been added, the mixture is incubated at 16°C for 1 hour or 16–18 hours. The recovered DNA is then diluted with reaction buffer again and recut by the same restriction enzyme.

The table below lists the ligation and recutting efficiencies of our restriction enzyme portfolio:

*Digested DNA was ligated using DNA Ligation Kit, Ver. 2.1 and incubated at 26°C for 10 min.

When using restriction enzymes to digest and analyze genomic DNA there may be many restriction enzymes available, depending on the species the genomic DNA was derived from. In order to offer only the most suitable restriction enzymes, we perform quality testing by cutting an appropriate bacterial genome DNA (agarose embedded; 0.5 µg DNA/50 µl gel) with each lot of an enzyme and then performing pulsed-field electrophoresis to confirm DNA band patterns.

Our quality assurance testing consists of adding 5, 10, 20, and 50 units of each enzyme and then incubating for 5 and 20 hours to determine the minimum amount of enzyme required for complete digestion.

The table below lists the minimum amount of restriction enzyme required for complete digestion of bacterial genomic DNA under various experimental conditions.

Product citations

Daniels, D. L. The complete AvrII restriction map of the Escherichia coli genome and comparisons of several laboratory strains. Nucleic Acids Res. 18, 2649–51 (1990).

McClelland, M., Jones, R., Patel, Y. & Nelson, M. Restriction endonucleases for pulsed field mapping of bacterial genomes. Nucleic Acids Res. 15, 5985–6005 (1987).

McClelland, M. & Nelson, M. Enhancement of the apparent cleavage specificities of restriction endonucleases: applications to megabase mapping of chromosomes. Gene Amplif. Anal. 5, 257–82 (1987).