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  • ‹ Back to Restriction enzyme overview
  • General information about restriction enzymes
  • Star activity of restriction enzymes
  • Inactivation of restriction enzymes
  • Buffer activity with restriction enzymes
  • Universal buffers for double digestion with restriction enzymes
  • Restriction enzymes affected by methylation
  • Methylation-sensitive restriction enzymes
  • QC of restriction enzymes
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Restriction enzyme overview
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Find a restriction enzyme
Home › Learning centers › Cloning › Traditional molecular cloning › Restriction enzyme overview › QC of restriction enzymes

Traditional molecular cloning

  • Restriction enzyme overview
    • General information about restriction enzymes
    • Star activity of restriction enzymes
    • Inactivation of restriction enzymes
    • Buffer activity with restriction enzymes
    • Universal buffers for double digestion with restriction enzymes
    • Restriction enzymes affected by methylation
    • Methylation-sensitive restriction enzymes
    • QC of restriction enzymes
  • Ligation cloning overview
  • Ligation product guide
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Restriction enzyme overview
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Restriction enzyme quality control

Each lot of restriction enzymes is assayed for quality based on the following experiments: 1) an overdigestion test where substrate DNA and excessive amounts of enzyme are incubated for 24 hours and any non-specific DNase is identified; 2) a pKF3 cloning test where extremely minute amounts of exonuclease can be identified; 3) a ligation-recutting efficiency test where any contamination from ligase inhibitors, phosphatases or exonucleases is determined; and 4) a genomic DNA analysis where restriction enzymes are added to the appropriate bacterial genomic DNA and normal DNA bands are confirmed after 24 hours' incubation.

Click on the following for more information about our QC tests:

pKF3 cloning test

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:

AccIII Aor13HI Aor51HI AvaII BalI
BamHI BanII BglII BspT104I BstPI
Bst1107I ClaI EcoO65I EcoRI EcoT14I
Eco52I FbaI HindIII HpaI KpnI
MluI NcoI NdeI NotI NsbI
PshBI PstI PvuI PvuII SacI
SacII SalI SmaI SphI Sse8387I
StuI VpaK11BI XbaI XhoI

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%

Ligation and recutting efficiency test

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:

Restriction enzymeLigation efficiency (%)Recutting efficiency (%)
AatII >90 >95
AccI >90 100
AccII >90 100
AccIII >95 100
AfaI 90 100
AflII >95 100
AluI >95 100
Aor13HI >95 100
Aor51HI 90 95
ApaI >95 100
ApaLI >95 100
AvaI >90 100
AvaII >90 100
BalI >90 100
BamHI >95 100
BanII 100 100
BcnI* 90 90
BglI >95 100
BglII >95 100
BlnI 90 100
Bpu1102I 90 100
BspT104I >95 100
BspT107I >95 100
Bsp1286I 90 100
Bsp1407I 90 100
BssHII 90 100
BstPI >95 100
BstXI >90 100
Bst1107I 90 90
Cfr10I 90 100
Cfr13I 90 100
ClaI >95 100
CpoI 100 100
Restriction enzymeLigation efficiency (%)Recutting efficiency (%)
DraI >90 100
EaeI 100 100
Eam1105I 90 90
EcoO65I 95 100
EcoO109I >95 100
EcoRI 100 100
EcoRV 90 100
EcoT14I 100 100
EcoT22I 90 100
Eco52I 90 100
Eco81I* 90 90
FbaI 100 100
FokI >95 100
FseI >95 100
HaeII >95 100
HaeIII >95 100
HapII >90 100
HhaI >90 100
HincII >95 100
HindIII 100 100
HinfI >90 100
HinlI 90 100
HpaI >95 100
KpnI >90 >95
MboI 95 100
MboII 90 100
MflI >95 100
MluI 100 100
MspI 90 100
MunI 90 100
MvaI* 90 90
NaeI 90 100
NcoI >95 100
Restriction enzymeLigation efficiency (%)Recutting efficiency (%)
NdeI 90 100
NheI 90 95
NotI >95 100
NruI 95 95
NsbI >90 >90
PmaCI 95 100
PshAI >90 100
PshBI 95 100
Psp1406I 90 100
PstI >95 100
PvuI >95 100
PvuII >95 100
SacI >99 100
SacII >90 95
SalI >95 100
Sau3AI >95 100
ScaI >95 100
SfiI 90 100
SmaI >95 100
SnaBI 90 >95
SpeI >99 100
SphI 100 100
Sse8387I 100 100
SspI 90 100
StuI >95 100
SwaI >95 >95
TaqI >90 >90
Tth111I >90 >90
Van91I 90 100
VpaK11BI >90 100
XbaI 100 100
XhoI >95 100
XspI >90 100

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

Genomic DNA analysis

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.

Genomic DNA for QCEnzyme amount required for complete digestion
Restriction
enzyme
Recognition
sequences*
Genomic DNA for QCReaction
buffer**
Reaction
temperature (°C)
Enzyme amount required
for complete digestion
Incubation
for 5 hr
Incubation
for 20 hr
AatII GACGTC Staphylococcus aureus T + BSA 37 50 5
Aor51HI AGCGCT Staphylococcus aureus M 37 50 20
BglI GCCNNNNNGGC Staphylococcus aureus Basal 37 10 5
BlnI CCTAGG Escherichia coli K 37 5 5
BspT104I TTCGAA Arthrobacter luteus L 37 40 5
BssHII GCGCGC Staphylococcus aureus M 50 5

5

Bst1107I GTATAC Arthrobacter luteus K 37 5 5
ClaI ATCGAT Arthrobacter luteus M 30 50 20
CpoI CGGWCCG Staphylococcus aureus K 30 5 5
DraI TTTAAA Pseudomonas aeruginosa M 37 100*** 100***
Eco52I CGGCCG Staphylococcus aureus Basal 37 10 5
MluI ACGCGT Staphylococcus aureus H 37 5 5
MunI CAATTG Arthrobacter luteus M + BSA 37 5

5

NaeI GCCGGC Staphylococcus aureus L 37 50 50
NheI GCTAGC Arthrobacter luteus M 37 50 20
NotI GCGGCCGC Escherichia coli H + BSA + Tri. 37 5 5
NruI TCGCGA Staphylococcus aureus Basal 37 50 5
NsbI TGCGCA Arthrobacter luteus T + BSA 37 5 5
PshBI ATTAAT Pseudomonas aeruginosa Basal 37 5 5
Psp1406I AACGTT Arthrobacter luteus T + BSA 37 5 5
PvuI CGATCG Flavobacterium okeanokoites K + BSA 37 20 5
SacII CCGCGG Staphylococcus aureus T + BSA 37 10 5
SalI GTCGAC Staphylococcus aureus H 37 150 5
SfiI GGCCNNNNNGGCC Escherichia coli M 50 100 50
SmaI CCCGGG Staphylococcus aureus T + BSA 30 5 5
SmiI ATTTAAAT Escherichia coli H 30 50 5
SnaBI TACGTA Arthrobacter luteus Basal 37 10 10
SpeI ACTAGT Escherichia coli M 37 50 20
Sse8387I CCTGCAGG Staphylococcus aureus M + BSA 37 5 5
SspI AATATT Arthrobacter luteus Basal 37 5 5
XbaI TCTAGA Escherichia coli M + BSA 37 50 5
XhoI CTCGAG Escherichia coli H 37 20 5


*W: A or T, N: A or C or G or T
**0.5 µg DNA/50 µl gel + 100 µl reaction mixture
***Reaction of DraI is extremely slow in agarose gel, so optimal amounts were added and left at 4°C for 16 hours. The samples were then transferred to 37°C and incubated for 5 or 20 hours.

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).

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