Recleavable Blunt Ends

New restriction sites can be generated by ligation of DNA fragments with compatible cohesive or blunt ends. These new restriction sites may be generated by:

1. Cleavage followed by fill-in of 5´ overhangs to generate blunt ends.
2. Cleavage with two restriction endonucleases that produce blunt ends.
3. Cleavage with two restriction endonucleases that produce compatible overhangs.

The table below lists combinations of blunt-end restriction endonucleases that produce recleavable ligation products.The combinations listed were identified by computer analysis, and although we have tried to ensure their accuracy, they have not necessarily been confirmed by experimentation.

Enzymes that have degenerate recognition sequences (i.e. recognize more than one sequence) are followed by a specific sequence in parentheses and are only listed if a non-degenerate equivalent does not exist. Be aware that these degenerate enzymes will cleave sequences in addition to the one listed. Where isoschizomers exist, only one member of each set is listed. Only commercially available enzymes are shown.

| A | B | D | E | F | H | M | N | P | R | S |    single letter code

Enzyme Ligated to Recleaved by
AfeI (AGC/GCT) BsrBI, MspA1I (CMG/CGG) HpaII
BstZ17I AluI
EcoRV SfaNI
SfoI HaeII, HhaI
FspI HhaI
NaeI AciI, Fnu4HI
SmaI AciI
AluI (AG/CT) BsrBI, MspA1I (CMG/CGG) AciI
BstZ17I RsaI
MspA1I (CMG/CTG), PvuII AluI
EcoRV MboI
SfoI HaeIII
BsaAI    
(YAC/GTR) BsrBI, MspA1I (CMG/CGG) HpaII
(CAC/GTR) PmlI BsaAI, PmlI
(TAC/GTR) PmlI, SnaBI BsaAI
(TAC/GTR) SnaBI BsaAI, SnaBI
BstUI (CG/CG) BsrBI, MspA1I (CMG/CGG) AciI, BstUI
BstZ17I RsaI
EcoRV MboI
SfoI HaeIII
NruI BstUI
BstZ17I (GTA/TAC) AluI, BsrBI, BstUI, HaeIII, MscI, MspA1I(CMG/CKG), NruI, PvuII, StuI RsaI
AfeI AluI
HincII (GTY/GAC) AccI
SspI Tsp509I
DraI (TTT/AAA) HincII (GTY/AAC), HpaI MseI
NruI TaqαI
PmeI, SwaI DraI, MseI
EcoRV *+
(GAT/ATC)
AluI, BsrBI, BstUI, MspA1I (CMG/CKG), PvuII MboI
HaeIII, MscI, StuI AlwI, MboI
AfeI, SfoI, FspI SfaNI
NruI MboI, TaqαI
FspI (TGC/GCA) BsrBI, MspA1I (CMG/CGG) HpaII
AfeI, SfoI HhaI
EcoRV SfaNI
NaeI AciI, Fnu4HI
SmaI AciI
HaeIII/PhoI (GG/CC) BsrBI, MspA1I (CMG/CGG) AciI
BstZ17I RsaI
MscI, StuI HaeIII
EcoRV AlwI, MboI
SfoI HaeIII, Asu96I
HincII (GTY/GAC) BsmFI
HpaI (GTT/AAC) DraI, PmeI MseI
HincII (GTY/AAC) HincII, HpaI, MseI
HincII (GTY/GAC) HincII
NruI TaqαI
HincII
(GTC/RAC) BsrBI, MspA1I (CMG/CGG) HpaII
(GTC/RAC) BstZ17I AccI
(GTC/RAC) HaeIII, MscI, StuI BsmFI
(GTT/RAC) DraI, PmeI MseI
(GTC/RAC) HpaI HincII
(GTT/RAC) HpaI HincII, HpaI, MseI
(GTT/RAC) NruI TaqαI
(GTC/RAC) RsaI, ScaI Tsp45I
MscI (TGG/CCA) BsrBI, MspA1I (CMG/CGG) AciI
BstZ17I RsaI
HaeIII, StuI HaeIII
EcoRV AlwI, MboI
SfoI HaeIII, Sau96I
HincII (GTY/GAC) BsmFI
MspA1I
(CAG/CKG) AluI AluI
(CCG/CKG) AluI, HaeIII, MscI, StuI AciI
(CCG/CKG) BsaAI, FspI, HincII (GTY/GAC), PmlI, SnaBI HpaII
(CAG/CKG) BsrBI, PvuII AciI, MspA1I
(CCG/CKG) BsrBI AciI, BsaJI, BstUI, MspA1I, SacII
(CMG/CKG) BstZ17I RsaI
(CCG/CKG) BstUI, NruI AciI, BstUI
(CCG/CKG) AfeI HpaII
(CMG/CKG) EcoRV MboI
(CMG/CKG) SfoI HaeIII
(CCG/CKG) SfoI HaeIII, HpaII
(CCG/CKG) NaeI HpaII, NciI, ScrFI
(CAG/CKG) PvuII AluI, MspA1I, PvuII
(CCG/CKG) SmaI BsaJI, HpaII, NciI, ScrFI
NaeI (GCC/GGC) BsrBI, MspA1I (CMG/CGG), SmaI HpaII, NciI, ScrFI
AfeI, SfoI, FspI AciI, Fnu4HI
NruI (TCG/CGA) BsrBI, MspA1I (CMG/CGG) AciI, BstUI
BstZ17I RsaI
BstUI BstUI
DraI, HincII (GTY/AAC), HpaI, PmeI, RsaI, ScaI, SspI TaqαI
EcoRV MboI, TaqαI
SfoI HaeIII
PmeI (GTTT/AAAC) DraI, SwaI DraI, MseI
HincII (GTY/AAC), HpaI MseI
NruI TaqαI
PmlI (CAC/GTG) BsaAI (YAC/GTA), SnaBI BsaAI
BsaAI (YAC/GTG) BsaAI, PmlI
BsrBI, MspA1I (CMG/CGG) HpaII
BmgBI PmlI
PvuII * (CAG/CTG) AluI AluI
BsrBI, MspA1I (CMG/CGG) AciI, MspA1I
Bstz17I RsaI
EcoRV MboI
SfoI HaeIII
MspA1I (CMG/CTG) AluI, MspA1I, PvuII
RsaI (GT/AC) HincII (GTY/GAC) Tsp45I
NruI TaqαI
ScaI RsaI
ScaI *+ (AGT/ACT) HincII (GTY/GAC) Tsp45I
NruI TaqαI
RsaI RsaI
SfoI (GGC/GCC) AluI, BstUI, MspA1I (CMG/CKG), NruI, PvuII HaeIII
BsrBI, MspA1I (CMG/CGG) HaeIII, HpaII
HaeIII, MscI, StuI HaeIII, Sau96I
AfeI HaeII, HhaI
EcoRV SfaNI
FspI HhaI
NaeI AciI, Fnu4HI
SmaI AciI
SmaI (CCC/GGG) BsrBI, MspA1I (CMG/CGG) BsaJI, HpaII, NciI, ScrFI
AfeI, SfoI, FspI AciI
NaeI HpaII, NciI, ScrFI
SnaBI (TAC/GTA) BsaAI (YAC/GTA) BsaAI, SnaBI
BsaAI (YAC/GTG), PmlI BsaAI
BsrBI, MspA1I (CMG/CGG) HpaII
SspI * (AAT/ATT) BstZ17I Tsp509I
NruI TaqαI
StuI (AGG/CCT) BsrBI, MspA1I (CMG/CGG) AciI
BstZ17I RsaI
HaeIII, MscI HaeIII
EcoRV AlwI, MboI
SfoI HaeIII, Sau96I
HincII (GTY/GAC) BsmFI
SwaI (ATTT/AAAT) DraI, PmeI DraI, MseI


* HF (high fidelity) versions of these enzymes are available for simplified reactions and reduced star activity, at no additional cost.

+ A RE-Mix version of this, or the HF version of this enzyme is available.