Reaction Conditions for Conventional Restriction Enzymes

The restriction enzyme reaction relies on precise conditions to achieve reliable, reproducible results. The table below provides detailed information on the optimal conditions for Thermo Fisher Scientific conventional restriction enzymes, including buffer compositions, reaction temperatures, and incubation times. Use the table as a restriction digest finder.

Easily determine enzymatic reaction buffer, enzyme concentrations, incubation conditions, and any additives needed in your double digest reaction using our DoubleDigest calculator

Restriction enzyme compatibility chart

Thermo Scientific enzyme	Recommended buffer for 100% activity	Enzyme activity in 5-buffer system buffers, %						Tango buffer for double digestion	Units for overnight incubation, u/μg DNA	Thermal inactivation in 20 min
Thermo Scientific enzyme	Recommended buffer for 100% activity	B (blue) 1X	G (green) 1X	O (orange) 1X	R (red) 1X	Tango (yellow) 1X / 2X		Tango buffer for double digestion	Units for overnight incubation, u/μg DNA	Thermal inactivation in 20 min
AanI (PsiI)	Tango	50-100	50-100	0-20	0-20	100	20-50	1X or 2X	0.5	65°C
AarI	AarI +oligo	NR (+oligo)	NR (+oligo)	0-20 (+oligo)	0-20 (+oligo)	NR (+oligo)	50-100 (+oligo)	2X (+oligo)	0.9	65°C
AatII	Tango	50-100	20-50	0-20	0-20	100	20-50	1X or 2X	0.3	65°C
Acc65I (Asp718I)	O	0-20	20-50	100	20-50	20-50	50-100	1X or 2X	0.3	65°C
AdeI (DraIII)	G	0-20	100	20-50	100	100*	20-50	1X* or 2X	0.5	No
AjiI (BmgBI)	AjiI	NR	NR	20-50*	NR	NR	20-50*	2X*	0.5	65°C
AjuI	R +SAM	0-20 (+SAM)	50-100 (+SAM)	20-50 (+SAM)	100 (+SAM)	50-100 (+SAM)	50-100 (+SAM)	1X or 2X (+SAM)	0.5	65°C
AloI	R	0-20	0-20	0-20	100	20-50	100	1X or 2X	0.1	65°C
AluI	Tango	50-100	0-20	0-20	0-20	100	20-50	1X or 2X	0.1	65°C
Alw21I (BsiHKAI)	O	0-20	20-50	100	50-100	20-50	50-100	1X or 2X	0.1	65°C
Alw26I	Tango	50-100	100	0-20	0-20	100	100	1X or 2X	0.2	65°C
Alw44I	Tango	50-100	100	0-20	50-100	100	50-100	1X or 2X	0.1	65°C
ApaI	B	100	20-50	0-20	0-20	20-50	0-20	1X	0.2	65°C
BamHI	BamHI	20-50*	100	20-50	50-100*	100*	50-100	1X* or 2X	0.5	80°C (10u)
BauI (BssSI)	Tango	0-20	50-100	0-20	50-100	100	50-100	1X or 2X	0.2	65°C
BclI	G	20-50	100	20-50	20-50	100*	100	1X* or 2X	0.1	80°C (10u)
BcnI (NciI)	Tango	20-50	50-100	50-100	50-100	100	50-100	1X or 2X	0.2	65°C
BcuI (SpeI)	Tango	50-100	50-100	0-20	20-50	100	0-20	1X	0.5	No
BfmI (SfcI)	Tango	0-20	50-100	0-20	0-20	100	20-50	1X or 2X	0.9	65°C
BfuI (BciVI)	BfuI	NR	NR	0-20	0-20	NR	50-100*	2X*	0.9	80°C
BglI	O	0-20	50-100	100	100	0-20	100	2X	0.1	65°C
BglII	O	0-20	20-50	100	50-100	0-20	100	2X	0.1	No
Bme1390I (ScrFI)	O	20-50	50-100	100	50-100	50-100	50-100	1X or 2X	0.2	80°C
BoxI (PshAI)	Tango	0-20	0-20	0-20	20-50	100	20-50	1X or 2X	0.5	80°C
BpiI (BbsI)	G	20-50	100	50-100	50-100	50-100	50-100	1X or 2X	0.3	65°C
BplI	Tango	0-20 (+SAM)	20-50 (+SAM)	0-20 (+SAM)	0-20 (+SAM)	100 (+SAM)	20-50 (+SAM)	1X (+SAM)	0.3	65°C
Bpu10I	Bpu10I	0-20	20-50*	50-100*	100*	50-100*	100*	1X* or 2X*	0.2	80°C
Bpu1102I (BlpI)	Tango	50-100	50-100	20-50	20-50	100	20-50	1X or 2X	0.1	80°C
BseDI (BsaJI)	Tango	50-100	20-50	0-20	0-20	100	50-100	1X or 2X	0.2	80°C
BseGI (BtsCI)	Tango	20-50	50-100	20-50	20-50	100	20-50	1X or 2X	0.2	80°C
BseJI (BsaBI)	O	NR	100*	100	NR	NR	100*	2X*	0.1	No
BseLI (BslI)	Tango	20-50	100	0-20	20-50	100	50-100	1X or 2X	0.1	No
BseMI (BsrDI)	R	0-20	20-50	0-20	100	50-100	50-100	1X or 2X	0.3	80°C
BseMII (BspCNI)	Tango	50-100 (+SAM)	50-100 (+SAM)	50-100 (+SAM)	50-100 (+SAM)	100 (+SAM)	50-100 (+SAM)	1X or 2X (+SAM)	0.5	80°C
BseNI (BsrI)	B	100	20-50	0-20	0-20	50-100	20-50	1X or 2X	0.1	80°C
BseSI (Bme1580I)	G	20-50	100	0-20	20-50	50-100	0-20	1X	0.1	80°C (10u)
BseXI (BbvI)	BseXI	NR	NR	NR	NR	NR	NR	NR	0.3	80°C
Bsh1236I (BstUI)	R	0-20	0-20	50-100	100	20-50	50-100	1X or 2X	0.1	65°C
Bsh1285I (BsiEI)	G	20-50	100	20-50	0-20	0-20	20-50	2X	0.2	65°C
BshNI (BanI)	O	0-20	20-50	100	50-100	0-20	100	2X	0.3	65°C
BshTI (AgeI)	O	0-20	20-50	100	50-100	20-50	20-50	1X or 2X	0.1	65°C
Bsp119I (BstBI)	Tango	20-50	0-20	0-20	0-20	100	100	1X or 2X	0.1	80°C
Bsp120I (PspOMI)	B	100	20-50	0-20	20-50	50-100	0-20	1X	0.1	80°C
Bsp1407I (BsrGI)	Tango	0-20	20-50	0-20	20-50	100	50-100	1X or 2X	0.1	65°C
Bsp143I (Sau3AI)	Bsp143I	20-50	20-50	0-20	0-20	50-100	20-50	1X or 2X	0.1	65°C
Bsp68I (NruI)	O	0-20	20-50	100	50-100	20-50	50-100	1X or 2X	0.1	65°C
BspLI (NlaIV)	Tango	50-100	50-100	0-20	20-50	100	20-50	1X or 2X	0.3	65°C
BspOI (BmtI)	O	0-20	0-20	100	100	0-20	20-50	2X	0.2	80°C
BspPI (AlwI)	Tango	20-50	20-50	0-20	0-20	100	0-20	1X	0.5	80°C
BspTI (AflII)	O	0-20	0-20	100	20-50	0-20	50-100	2X	0.1	65°C
Bst1107I (BstZ17I)	O	20-50	50-100	100	100	20-50	100	1X or 2X	0.1	65°C
BstXI	O	20-50	100	100	50-100	50-100	100	1X or 2X	0.1	65°C
Bsu15I (ClaI)	Tango	20-50	20-50	20-50	20-50	100	20-50	1X or 2X	0.1	65°C
BsuRI (HaeIII)	R	0-20	0-20	0-20	100	50-100	100	1X or 2X	0.1	80°C
BveI (BspMI)	O +oligo	0-20 (+oligo)	20-50 (+oligo)	100 (+oligo)	20-50 (+oligo)	50-100 (+oligo)	100 (+oligo)	1X or 2X (+oligo)	0.2	65°C
CaiI (AlwNI)	Tango	20-50	20-50	20-50	50-100	100	50-100	1X or 2X	0.2	65°C
Cfr10I (BsrFI)	Cfr10I	0-20	20-50	20-50	50-100	20-50	50-100	1X or 2X	0.1	No
Cfr13I (Sau96I)	Tango	50-100	50-100	20-50	20-50	100	20-50	1X or 2X	0.3	65°C
Cfr42I (SacII)	B	100	50-100	0-20	0-20	50-100	0-20	1X	0.1	65°C
Cfr9I (XmaI)	Cfr9I	0-20	0-20	0-20	0-20	20-50	0-20	1X	0.2	65°C
CpoI (RsrII)	Tango	20-50	50-100	50-100	20-50	100	50-100	1X or 2X	0.5	65°C
CseI (HgaI)	R	NR	50-100*	50-100	100	100*	50-100	1X* or 2X	0.5	80°C (10u)
Csp6I (CviQI)	B	100	50-100	0-20	0-20	50-100	0-20	1X	0.1	65°C
DpnI	Tango	100	100	50-100	50-100	100	50-100	1X or 2X	0.1	80°C
DraI	Tango	50-100	50-100	20-50	20-50	100	50-100	1X or 2X	0.1	65°C
Eam1104I (EarI)	Tango	50-100	50-100	0-20	0-20	100	0-20	1X	0.5	65°C
Eam1105I (AhdI)	Eam1105I	20-50	50-100	0-20	0-20	50-100	20-50	1X or 2X	0.1	65°C
Ecl136II (EcoICRI)	Ecl136II	50-100	20-50	0-20	0-20	50-100	0-20	1X	0.2	65°C
Eco105I (SnaBI)	Tango	100*	50-100	0-20	0-20	100	0-20	1X	0.5	65°C
Eco130I (StyI)	O	0-20	20-50	100	50-100	50-100	100	1X or 2X	0.2	65°C
Eco147I (StuI)	B	100	50-100	20-50	20-50	50-100	0-20	1X	0.1	80°C
Eco24I (BanII)	Tango	50-100	50-100	20-50	20-50	100	0-20	1X	0.2	65°C
Eco31I (BsaI)	G	50-100	100	0-20	0-20	50-100	20-50	1X or 2X	0.3	65°C
Eco32I (EcoRV)	R	0-20	50-100	50-100	100	20-50	100	1X or 2X	0.1	65°C
Eco47I (AvaII)	R	0-20	50-100	50-100	100	50-100	50-100	1X or 2X	0.3	65°C
Eco47III (AfeI)	O	0-20	20-50	100	100	50-100	100	1X or 2X	0.1	80°C
Eco52I (EagI)	Eco52I	0-20	0-20	0-20	20-50	0-20	20-50	2X	0.2	65°C
Eco57I (AcuI)	G +SAM	100 (+SAM)	100 (+SAM)	20-50 (+SAM)	20-50 (+SAM)	50-100 (+SAM)	50-100 (+SAM)	1X or 2X (+SAM)	0.9	65°C
Eco72I (PmlI)	Tango	NR	NR	0-20	0-20	100	20-50	1X or 2X	0.5	65°C
Eco81I (Bsu36I)	Tango	50-100	100	0-20	0-20	100	0-20	1X	0.1	80°C
Eco88I (AvaI)	Tango	100	50-100	0-20	0-20	100	20-50	1X or 2X	0.2	65°C
Eco91I (BstEII)	O	20-50	20-50	100	50-100	NR	100	2X	0.1	65°C
EcoO109I (DraII)	Tango	50-100	20-50	20-50	20-50	100	100	1X or 2X	0.2	65°C
EcoRI	EcoRI	0-20	NR	100	100*	NR	100	2X	0.2	65°C
EcoRII	O	20-50	50-100	100	50-100	20-50	50-100	1X or 2X	0.1	80°C
EheI (SfoI)	Tango	20-50	50-100	0-20	0-20	100	20-50	1X or 2X	0.9	65°C
Esp3I (BsmBI)	Tango	100 (+DTT)	20-50 (+DTT)	0-20 (+DTT)	0-20 (+DTT)	100 (+DDT)	0-20 (+DTT)	1X (+DDT)	0.2	65°C
FaqI (BsmFI)	Tango	20-50 (+SAM)	20-50 (+SAM)	0-20 (+SAM)	0-20 (+SAM)	100 (+SAM)	50-100 (+SAM)	1X (+SAM)	0.9	80°C
FspAI	O	0-20	0-20	100	50-100	0-20	50-100	2X	0.2	65°C
FspBI (BfaI)	Tango	50-100	20-50	0-20	0-20	100	0-20	1X	0.3	65°C
GsuI (BpmI)	B	100	50-100	0-20	20-50	100	50-100	1X or 2X	0.9	65°C
HhaI	Tango	50-100	50-100	20-50	20-50	100	20-50	1X or 2X	0.1	80°C
Hin1I (BsaHI)	G	20-50	100	20-50	20-50	20-50	20-50	1X or 2X	0.1	65°C
Hin1II (NlaIII)	G	50-100	100	20-50	50-100	50-100	50-100	1X or 2X	0.3	65°C
Hin6I (HinP1I)	Tango	50-100	50-100	20-50	20-50	100	50-100	1X or 2X	0.1	65°C
HincII (HindII)	Tango	50-100	50-100	20-50	50-100	100	50-100	1X or 2X	0.1	65°C
HindIII	R	0-20	20-50	0-20	100	50-100	50-100	1X or 2X	0.1	80°C
HinfI	R	0-20	20-50	50-100	100	50-100	50-100	1X or 2X	0.1	65°C
HpaII	Tango	50-100	50-100	0-20	20-50	100	20-50	1X or 2X	0.1	65°C
HphI	B	100	0-20	0-20	0-20	20-50	0-20	1X	0.1	65°C
Hpy8I (MjaIV)	Tango	50-100	50-100	0-20	20-50	100	50-100	1X or 2X	0.1	80°C
HpyF10VI (MwoI)	Tango	0-20	0-20	0-20	0-20	100	50-100	1X or 2X	0.1	80°C
HpyF3I (DdeI)	Tango	20-50	20-50	20-50	20-50	100	50-100	1X or 2X	0.2	65°C
I-SceI	Tango	50-100	50-100	50-100	50-100	100	50-100	1X or 2X	0.9	65°C
Kpn2I (BspEI)	Tango	50-100	50-100	0-20	20-50	100	50-100	1X or 2X	0.1	80°C
KpnI	KpnI	20-50	0-20	0-20	0-20	20-50	0-20	1X	0.2	80°C
KspAI (HpaI)	B	100	50-100*	20-50	20-50	100*	50-100	1X* or 2X*	0.5	65°C
LguI (SapI)	Tango	20-50	50-100	20-50	20-50	100	20-50	1X or 2X	0.5	65°C
Lsp1109I (BbvI)	Lsp1109I	0-20	20-50*	50-100*	100	20-50*	20-50*	1X* or 2X	0.5	65°C
LweI (SfaNI)	Tango	0-20	0-20	0-20	20-50	100	20-50	1X or 2X	0.2	65°C
MauBI	Tango	0-20	0-20	0-20	0-20	100	0-20	1X	0.1	65°C
MbiI (BsrBI)	Tango	20-50	100	0-20	20-50	100	20-50	1X or 2X	0.2	65°C
MboI	R	50-100	50-100	50-100	100	50-100	100	1X or 2X	0.1	65°C
MboII	B	100	50-100	20-50	0-20	50-100	20-50	1X or 2X	0.9	65°C
MlsI (MscI)	R	0-20	20-50	0-20	100	20-50	50-100	1X or 2X	0.5	65°C
MluI	R	0-20	20-50	50-100	100	20-50	50-100	1X or 2X	0.1	65°C
MnlI	G	50-100	100	20-50	20-50	20-50	20-50	1X or 2X	0.5	65°C
Mph1103I (NsiI)	R	0-20	50-100	20-50	100	50-100	50-100	1X or 2X	0.3	65°C
MreI (Sse232I)	G	0-20	100	0-20	0-20	50-100	0-20	1X	0.5	80°C
MspI (HpaII)	Tango	50-100	50-100	0-20	0-20	100	50-100	1X or 2X	0.3	65°C
MssI (PmeI)	B	100	0-20	0-20	0-20	20-50	0-20	1X	0.5	65°C
MunI (MfeI)	G	100	100	0-20	0-20	100	0-20	1X	0.1	65°C
Mva1269I (BsmI)	R	0-20	20-50	50-100	100	0-20	50-100	2X	0.1	65°C
MvaI (BstNI)	R	20-50	20-50	50-100	100	20-50*	100	1X* or 2X	0.1	80°C (10u)
Nb.Bpu10I	R	0-20	20-50	20-50	100	20-50	50-100	1X or 2X	0.3	80°C
Nb.Mva1269I	O	0-20	20-50	100	100	20-50	50-100	1X or 2X	0.3	80°C
NcoI	Tango	20-50	20-50	20-50	50-100	100	100	1X or 2X	0.1	65°C
NdeI	O	0-20	0-20	100	50-100	0-20	50-100	2X	0.3	65°C
NheI	Tango	100	20-50	0-20	0-20	100	0-20	1X	0.2	65°C
NmuCI (Tsp45I)	R	0-20	20-50	50-100	100	20-50	50-100	1X or 2X	0.1	65°C
NotI	O	0-20	20-50	100	20-50	0-20	20-50	2X	0.1	65°C
NsbI (FspI)	Tango	20-50	50-100	0-20	20-50	100	20-50	1X or 2X	0.1	65°C
Nt.Bpu10I	R	0-20	0-20	100	100	0-20	50-100	2X	0.3	80°C
OliI (AleI)	R	0-20	0-20	0-20	100	0-20	50-100	2X	0.2	65°C
PacI	PacI	20-50	20-50	0-20	0-20	0-20	0-20	NR	0.3	65°C
PaeI (SphI)	B	100	50-100	0-20	0-20	50-100	0-20	1X	0.1	65°C
PagI (BspHI)	O	0-20	50-100	100	NR	NR	NR	NR	0.2	80°C
PasI	PasI	NR	NR	NR	NR	NR	NR	NR	0.2	80°C
PauI (BssHII)	R	0-20	0-20	100	100	0-20	100	2X	0.2	80°C
PdiI (NaeI)	Tango	50-100	20-50	0-20	0-20	100	50-100	1X or 2X	0.5	65°C
PdmI (XmnI)	Tango	20-50	50-100	0-20	0-20	100	0-20	1X	0.5	65°C
PfeI (TfiI)	O	0-20	20-50	100	50-100	20-50	50-100	1X or 2X	0.1	65°C
Pfl23II (BsiWI)	Tango	20-50	50-100	20-50	20-50	100	0-20	1X	0.3	65°C
PfoI	Tango	0-20	20-50	50-100	0-20	100	50-100	1X or 2X	0.1	65°C
Ppu21I (BsaAI)	Ppu21I	50-100*	100*	20-50	NR	NR	NR	NR	0.5	65°C
PscI (PciI)	Tango	20-50	20-50	0-20	0-20	100	0-20	1X	0.2	65°C
Psp1406I (AclI)	Tango	100	50-100	0-20	20-50	100	0-20	1X	0.5	65°C
Psp5II (PpuMI)	G	0-20	100	20-50	20-50	50-100	100	1X or 2X	0.2	80°C
PstI	O	50-100	50-100	100	100	50-100	50-100	1X or 2X	0.2	80°C (10u)
PsuI (BstYI)	B	100	20-50	0-20	0-20	50-100	0-20	1X	0.5	80°C
PsyI (Tth111I)	B	100	50-100	0-20	0-20	50-100	0-20	1X	0.1	80°C
PvuI	R	0-20	20-50	50-100	100	50-100	100	1X or 2X	0.2	80°C (10u)
PvuII	G	50-100*	100	20-50	50-100	20-50*	20-50*	1X* or 2X*	0.2	80°C
RsaI	Tango	50-100	20-50	0-20	0-20	100	0-20	1X	0.2	65°C
RseI (MslI)	R	0-20	50-100	50-100	100	20-50	100	1X or 2X	0.5	65°C
SacI	SacI	50-100	20-50	0-20	0-20	50-100	20-50	1X or 2X	0.2	65°C
SalI	O	0-20	0-20	100	20-50	0-20	50-100	2X	0.1	65°C
SatI (Fnu4HI)	G	20-50	100	20-50	20-50	50-100	20-50	1X or 2X	0.1	65°C
ScaI	ScaI	0-20	0-20	0-20	0-20	0-20	0-20	NR	0.3	80°C (10u)
SchI (MlyI)	Tango	20-50	50-100	0-20	0-20	100	0-20	1X	0.2	65°C
SdaI (SbfI)	SdaI	NR	NR	0-20	0-20	NR	20-50	2X	0.3	65°C
SduI (Bsp1286I)	SduI	NR	50-100*	0-20	0-20	NR	NR	NR	0.3	65°C
SfaAI (AsiSI)	Tango	50-100	0-20	0-20	0-20	100	0-20	1X	0.2	80°C
SfiI	G	50-100	100	20-50	0-20	100	0-20	1X	0.2	80°C
SgeI	SgeI	0-20	0-20	0-20	NR	NR	NR	NR	0.0	65°C
SgrDI	R	0-20	0-20	0-20	100	NR	100	2X	0.3	65°C
SgsI (AscI)	Tango	0-20	0-20	0-20	50-100	100	50-100	1X or 2X	0.1	65°C
SmaI	Tango	50-100	0-20	0-20	0-20	100	0-20	1X	0.2	65°C
SmiI (SwaI)	O	0-20	0-20	100	20-50	0-20	20-50	2X	0.1	65°C
SmoI (SmlI)	Tango	50-100	20-50	0-20	20-50	100	20-50	1X or 2X	0.2	80°C
SsiI (AciI)	O	NR	20-50	100	50-100	NR	100	2X	0.5	65°C
SspDI (KasI)	Tango	20-50	0-20	NR	0-20	100	20-50	1X or 2X	1.0	80°C
SspI	G	20-50	100	0-20	50-100	100	20-50	1X or 2X	0.1	65°C
TaaI (HpyCH4III)	Tango	0-20	0-20	0-20	50-100	100	100	1X or 2X	0.2	No
TaiI (MaeII)	R	50-100	50-100	20-50	100	100	50-100	1X or 2X	0.3	No
TaqI	TaqI	0-20	20-50	20-50	20-50	20-50	20-50	1X or 2X	0.3	No
TasI (Tsp509I)	B	100	50-100	20-50	0-20	20-50	0-20	1X	0.3	No
TatI	Tango	NR	50-100*	0-20	20-50	100*	0-20	1X*	0.2	No
TauI	B	100	50-100	0-20	0-20	20-50	0-20	1X	0.9	No
Tru1I (MseI)	R	50-100	50-100	20-50	100	50-100	100	1X or 2X	0.2	No
TscAI (TspRI)	Tango	50-100	50-100	20-50	20-50	100	20-50	1X or 2X	0.2	No
Van91I (PflMI)	R	0-20	50-100	50-100	100	20-50	50-100	1X or 2X	0.1	65°C
VspI (AseI)	O	0-20	50-100	100	20-50	100	100	1X or 2X	0.1	65°C
XagI (EcoNI)	R	0-20	20-50	50-100	100	20-50	50-100	1X or 2X	0.1	65°C
XapI (ApoI)	Tango	50-100	100	0-20	0-20	100	20-50	1X or 2X	0.1	65°C
XbaI	Tango	50-100	50-100	20-50	0-20	100	50-100	1X or 2X	0.1	65°C
XceI (NspI)	Tango	50-100	0-20	0-20	0-20	100	0-20	1X	0.2	65°C
XhoI	R	0-20	50-100	50-100	100	20-50	100	1X or 2X	0.1	80°C
XmaJI (AvrII)	Tango	20-50	50-100	50-100	50-100	100	50-100	1X or 2X	0.2	80°C
XmiI (AccI)	B	100	0-20	0-20	0-20	50-100	20-50	1X or 2X	0.1	65°C

Resources & support for restriction enzymes

Conventional Restriction Enzymes
FastDigest Restriction Enzymes
Cloning Learning Center

For Research Use Only. Not for use in diagnostic procedures.

Stylesheet for Classic Wide Template adjustments

Reaction Conditions for Conventional Restriction Enzymes

Restriction enzyme compatibility chart

Products related to restriction enzymes

Tools to help your restriction digest

Educational articles