Restriction enzymes requiring multi-sites for efficient cleavage

This table indicates which NEB restriction enzymes have been identified to require binding at more than one recognition site for efficient cleavage. These enzymes are indicated with the icon 2site. Please visit "Restriction Enzyme Cleavage: ‘single-site’ enzymes and ‘multi-site’ enzymes” for more information.

RECOMMENDATIONS

If you are using an enzyme that may require more than one recognition site on the substrate to cleave optimally, we suggest two possible optimization methods:

  1. Titrate the units of enzyme used in the reaction to determine the optimal enzyme to substrate ratio. As a starting point, we recommend using 1-2 μl of restriction enzyme (at the supplied units/μl) per microgram of substrate and performing 2-fold serial dilutions of the enzyme, keeping the DNA concentration constant.
  2. If that is not possible, add duplex oligonucleotides that contain the recognition site. The oligos provide the additional recognition site needed to activate substrate-bound, but dormant enzyme monomers. Addition of duplex DNA containing a recognition site can compete for binding and reduce the effective enzyme concentration. To use oligos effectively, the stoichiometry of the reaction needs to be established beforehand by performing a series of titrations and identifying the optimum range for the concentration of oligos. We recommend keeping the enzyme concentration constant at 2-4 fold above the optimum established in step 1, above, while performing 2-fold serial dilutions of the oligo. As a starting point, we recommend a ratio of 4:1 (oligo sites:substrate sites) and performing a 2-fold serial dilution of the oligo, keeping the enzyme and substrate concentrations constant.

Enzyme Cut Site
BbvI GCAGC(8/12)
BcgI (10/12)CGANNNNNNTGC(12/10)
BfuAI ACCTGC(4/8)
BpmI CTGGAG(16/14)
BsgI GTGCAG(16/14)
BspMI ACCTGC(4/8)
CspCI (11/13)CAANNNNNGTGG(12/10)
EcoP15I CAGCAG(25/27)
FokI GGATG(9/13)
MboII GAAGA(8/7)
MmeI TCCRAC(20/18)
NaeI GCC/GGC
NarI GG/CGCC
NgoMIV G/CCGGC
NmeAIII GCCGAG(21/19)
PleI GAGTC(4/5)
PluTI GGCGC/C
RsrII CG/GWCCG
SacII CCGC/GG
SfiI GGCCNNNN/NGGCC
SgrAI CR/CCGGYG
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