Cleavage Close to the End of DNA Fragments

Annealed 5´ FAM labeled oligos were incubated with the indicated enzyme (10 units/ 1pmol oligo) for 60 minutes at the recommended incubation temperature and NEBuffer. The digest was run on a TBE acrylamide gel and analyzed by fluorescent imaging. The double stranded oligos were designed to have the indicated number of base pairs from the end followed by the recognition sequence and an additional 12 bases. In some cases asymmetric cleavage was observed and interpreted as a negative result. Asymmetric cleavage decreased with increasing base pairs from the end.

Note: As a general rule and for enzymes not listed below, 6 base pairs should be added on on either side of the recognition site to cleave efficiently. The extra bases should be chosen so that palindromes and primer dimers are not formed. In most cases there is no requirement for specific bases.

Note: You may download the previous tables, which use a different methodology here:
Cleavage Close to the End of DNA Fragments (oligonucleotides) and
Cleavage Close to the End of DNA Fragments (linearized vector)
  1. Restriction Enzyme Digest Protocol: Cutting Close to DNA End

    When cutting close to the end of a DNA molecule, make sure you know how many bases to add to the ends of your PCR primers.

  2. Why is My Restriction Enzyme Not Cutting DNA?

    Not getting the cleavage you expected? Let an NEB scientist help you troubleshoot your reaction.

  3. What are Restriction Enzymes?

    Watch as Geoff Wilson, Restriction Enzymes Division Head, describes what restriction enzymes are and how they revolutionized molecular biology.

Chart Legend
 
0% + 0-20%
++ 20-50% +++ 50-100%
nt not tested  
EnzymeBase Pairs from end
1 bp2 bp3 bp4 bp5 bp
AciI-+++++++
AgeI+++++++++++++++
AgeI-HF®++++++++++++++
AluI-++++++++++++
ApaI+++++++++++++++
AscI+++++++++++++++
AvrII+++++++++++++
EnzymeBack to topBase Pairs from end
1 bp2 bp3 bp4 bp5 bp
BamHI++++++++++++
BamHI-HF®+++++++++++
BbsI-HF®+++++++++++++++
BglII++++++++++++++
BmtI+++++++++++++++
BmtI-HF®+++++++++++++++
BsaI+++++++++++++++
BsaI-HF®+++++++++++++++
BsiWI++++++++++++++
BsiWI-HF®+++++++++++++++
BsmBI+++++++++++++++
BsrGI+++++++++++++++
BssHII+++++++++++++
EnzymeBase Pairs from end
1 bp2 bp3 bp4 bp5 bp
BstZ17I-HF®+++++++++++++
EnzymeBack to topBase Pairs from end
1 bp2 bp3 bp4 bp5 bp
ClaI--+++++++
EnzymeBack to topBase Pairs from end
1 bp2 bp3 bp4 bp5 bp
DdeI+++++++++++++++
DpnI-++++ntnt
DraIII+++++++++++++++
DraIII-HF®+++++++++++++++
EnzymeBack to topBase Pairs from end
1 bp2 bp3 bp4 bp5 bp
EagI++++++++++++++
EagI-HF®+++++++++++++
EcoRI+++++++++
EcoRI-HF®++++++++++
EcoRV+++++++++++
EcoRV-HF®++++++++++
EnzymeBack to topBase Pairs from end
1 bp2 bp3 bp4 bp5 bp
FseI++++++++++++
EnzymeBack to topBase Pairs from end
1 bp2 bp3 bp4 bp5 bp
HindIII-++++++++++
HindIII-HF®-++++++++++
HpaI+++++++++++++++
EnzymeBack to topBase Pairs from end
1 bp2 bp3 bp4 bp5 bp
KpnI+++++++++++++
KpnI-HF®+++++++++++++
EnzymeBack to topBase Pairs from end
1 bp2 bp3 bp4 bp5 bp
MfeI++++++++++++
MfeI-HF®++++++++++++
EnzymeBase Pairs from end
1 bp2 bp3 bp4 bp5 bp
MluI++++++++++++
MseI+++++++++++++++
EnzymeBack to topBase Pairs from end
1 bp2 bp3 bp4 bp5 bp
NcoI-+++++++++++
NcoI-HF®++++++++++++
NdeI+++++++++++
NheI++++++++++++
NheI-HF®+++++++++++++
NlaIII++++++++++++++
NotI++++++++++
NotI-HF®++++++++++
NsiI+++++++++++
NspI--+++++
EnzymeBack to topBase Pairs from end
1 bp2 bp3 bp4 bp5 bp
PacI+++++++++++++++
PciI+++++++++++++++
PmeI+++++++++++++++
PstI+++++++++++++
PstI-HF®++++++++++++++
PvuI+++++++++++++++
PvuI-HF®+++++++++++++++
PvuII++++++++++++
EnzymeBase Pairs from end
1 bp2 bp3 bp4 bp5 bp
PvuII-HF®-++++++++++
EnzymeBack to topBase Pairs from end
1 bp2 bp3 bp4 bp5 bp
RsaI+++++++++++++
EnzymeBack to topBase Pairs from end
1 bp2 bp3 bp4 bp5 bp
SacI-+++++++++++
SacI-HF®-++++++++++
SacII+++++++++++++++
SalI-+++++++++++
SalI-HF®-+++++++++++
SapI+++++++++++++++
Sau3AI+++++++++++++++
SbfI++++++++++++++
SbfI-HF®++++++++++++++
ScaI+++++++++++++++
ScaI-HF®+++++++++++++
SfiI+++++++++++++++
SmaI+++++++++++++++
SpeI+++++++++
SpeI-HF®+++++++++
SphI+++++++++++++++
SphI-HF®+++++++++++++
SspI+++++++++++++
EnzymeBase Pairs from end
1 bp2 bp3 bp4 bp5 bp
SspI-HF®+++++++++++++
StuI+++++++++++++++
StyI++++++++++++
StyI-HF®+++++++++++++
EnzymeBack to topBase Pairs from end
1 bp2 bp3 bp4 bp5 bp
XbaI++++++++++
XhoI++++++++++++
XmaI+++++++++++++++
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