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How do I Perform Double Digest?

• DoubleDigest™ Engine
• Chart: "Reaction Conditions for Restriction Enzymes" (see below)
• Chart: Double Digestions using Universal Tango™ Buffer"

1. DoubleDigest™ Engine

Simply select two restriction enzymes, submit the query and read the recommendations.

2. Chart: "Reaction Conditions for Restriction Enzymes"

This table presents the relative activity (% of the activity in the optimal buffer) of the Fermentas restriction enzymes in the Five Buffer System.

1. Determine which color-coded buffers are recommended for each enzyme.
2. If the recommended color-coded buffer for both enzymes is the same, use that buffer.
3. If such a buffer is not indicated, choose the buffer in which both enzymes maintain at least 20% of their activity. Increase the amount of the enzymes in your digest according to their activity in that buffer.

Note
For enzymes that are prone to relaxation of specificities, use a buffer in which they do not exhibit star activity.

Note
To achieve effective digestion with both mesophilic and thermophilic enzymes (e.g., SmaI, TaaI), we recommend DNA digestion at the lower temperature first, and then increase the digestion temperature. The optimal reaction temperature for each restriction enzyme is indicated both in the product description and in the Certificate of Analysis supplied with each enzyme. Information about activity of thermophilic restriction enzymes at 37°C is presented here.

Sequential Digestion. If neither buffer is compatible with both restriction enzymes due to low enzyme activity (lower than 20%) or to the star activity, then perform sequential digestions in buffers optimal for each enzyme.

1. Digest the DNA with the first restriction enzyme in its optimal buffer.
2. Purify the digested DNA by phenol/chloroform extraction and ethanol precipitation (see protocol).
3. Digest the DNA with the second restriction enzyme in its optimal buffer.

3. Chart: Double Digestions using Universal Tango™ Buffer

Use the table below to set up optimal conditions for your double digest in the universal Tango™ buffer.

1. Determine the concentration of universal Tango™ buffer recommended for each restriction enzyme.
2. If the same Tango™ buffer concentration is recommended for both enzymes, use it.
3. If the two restriction enzymes require different Tango™ buffer concentrations, perform the first digestion with the enzyme recommended for the 1X Tango™ buffer. After digestion, add an additional aliquot of the 10X Tango™ buffer (1/8 of initial reaction volume) to get 2X Tango™ buffer. Then, digest DNA with the second enzyme.

Note
If both the 1X and the 2X concentrations of Tango™ buffer are suitable for double digestion, use the 2X concentrated buffer to avoid star activity.

A   B   CD   EF   GHKL   MNO   PR   STVX

Note
Yellow shaded buffers yield 50-100% and grey shaded buffers yield 20-50% of restriction enzyme activity.
NR: Buffer is not recommended, because of high star activity.
* Star activity appears at 5-fold overdigestion (5 units x 1 hour).
Optimal temperature of enzymes enlisted is 37°C, unless otherwise indicated in parenthesis.

A

Fermentas enzyme	Buffers for double digestions
	Optimal buffer	Tango™ 1X	Tango™ 2X
AanI	Tango™
AarI	AarI+oligo	NR	+oligo
AasI	B	*	NR
AatII	Tango™
Acc65I	O
AdeI	G	*
AjiI	AjiI	NR	*
AjuI	R+SAM	+SAM	+SAM
AlfI	R+SAM	NR	+SAM
AloI (30°C)	R
AluI	Tango™
Alw21I	O
Alw26I	Tango™
Alw44I	Tango™
ApaI	B		NR

B

Fermentas enzyme	Buffers for double digestions
	Optimal buffer	Tango™ 1X	Tango™ 2X
BamHI	BamHI	*
BauI	Tango™
BclI (55°C)	G	*
BcnI	Tango™
BcuI	Tango™		NR
BdaI (30°C)	G+SAM	+SAM*	+SAM
BfiI	Tango™		NR
BfmI	Tango™
BfuI	BfuI	NR	*
BglI	O	NR
BglII	O	NR
Bme1390I	O
BoxI	Tango™
BpiI	G
BplI	Tango™+SAM	+SAM	NR
Bpu10I	Bpu10I	*	*
Bpu1102I	Tango™
BseDI (55°C)	Tango™
BseGI (55°C)	Tango™
BseJI (65°C)	O	NR	*
BseLI (55°C)	Tango™
BseMI (55°C)	R
BseMII (55°C)	Tango™+SAM	+SAM	+SAM
BseNI (65°C)	B
BseSI (55°C)	G		NR
BseXI (65°C)	BseXI	NR	NR
Bsh1236I	R
Bsh1285I	G	NR
BshNI	O	NR
BshTI	O
Bsp68I	O
Bsp119I	Tango™
Bsp120I	B		NR
Bsp143I	Bsp143I
Bsp1407I	Tango™
BspLI	Tango™
BspPI (55°C)	Tango™		NR
BspTI	O	NR
Bst1107I	O
BstXI (55°C)	O
Bsu15I	Tango™
BsuRI	R
BveI	O+oligo	+oligo	+oligo

CD

Fermentas enzyme	Buffers for double digestions
	Optimal buffer	Tango™ 1X	Tango™ 2X
CaiI	Tango™
CfrI	Tango™		NR
Cfr9I	Cfr9I		NR
Cfr10I	Cfr10I
Cfr13I	Tango™
Cfr42I	B		NR
CpoI	Tango™
CseI	R	*
Csp6I	B		NR
DraI	Tango™
DpnI	Tango™

EF

Fermentas enzyme	Buffers for double digestions
	Optimal buffer	Tango™ 1X	Tango™ 2X
Eam1104I	Tango™		NR
Eam1105I	Eam1105I
Ecl136II	Ecl136II		NR
Eco24I	Tango™		NR
Eco31I	G
Eco32I	R
Eco47I	R
Eco47III	O
Eco52I	Eco52I	NR
Eco57I	G+SAM	+SAM	+SAM
Eco57MI	B+SAM	+SAM	NR
Eco72I	Tango™
Eco81I	Tango™		NR
Eco88I	Tango™
Eco91I	O
Eco105I	Tango™		NR
Eco130I	O
Eco147I	B		NR
EcoO109I	Tango™
EcoRI	EcoRI	NR
EcoRII	O
EheI	Tango™
Esp3I	Tango™+DTT	+DTT	NR
FaqI	Tango™+SAM	+SAM	+SAM
FspAI	O	NR
FspBI	Tango™		NR

GHKL

Fermentas enzyme	Buffers for double digestions
	Optimal buffer	Tango™ 1X	Tango™ 2X
GsuI (30°C)	B
HhaI	Tango™
Hin1I	G
Hin1II	G
Hin4I	Tango™+SAM	+SAM	NR
Hin6I	Tango™
HincII	Tango™
HindIII	R
HinfI	R
HpaII	Tango™
HphI	B		NR
Hpy8I	Tango™
HpyF3I	Tango™
HpyF10VI	Tango™
KpnI	KpnI		NR
Kpn2I (55°C)	Tango™
KspAI	B	*
LguI	Tango™
Lsp1109I	Lsp1109I	*	*
LweI	Tango™

MNO

Fermentas enzyme	Buffers for double digestions
	Optimal buffer	Tango™ 1X	Tango™ 2X
MauBI	Tango™	NR	NR
MbiI	Tango™
MboI	R
MboII	B
MlsI	R
MluI	R
MnlI	G
Mph1103I	R
MspI	Tango™
MssI	B		NR
MunI	G		NR
MvaI	R	*
Mva1269I	R	NR
NcoI	Tango™
NdeI	O	NR
NheI	Tango™		NR
NmuCI	R
NotI	O	NR
NsbI	Tango™
OliI	R	NR

PR

Fermentas enzyme	Buffers for double digestions
	Optimal buffer	Tango™ 1X	Tango™ 2X
PaeI	B		NR
PagI	O	NR	NR
PasI (55°C)	PasI	NR	NR
PauI	R	NR
PdiI	Tango™
PdmI	Tango™		NR
Pfl23II	Tango™		NR
PfeI	O
PfoI	Tango™
PpiI (55°C)	R
Ppu21I (30°C)	Ppu21I	NR	NR
PscI	Tango™		NR
Psp5II	G
Psp1406I	Tango™		NR
PstI	O
PsuI	B		NR
PsyI	B		NR
PvuI	R
PvuII	G	*	*
RsaI	Tango™		NR
RseI	R

STVX

Fermentas enzyme	Buffers for double digestions
	Optimal buffer	Tango™ 1X	Tango™ 2X
SacI	SacI
SalI	O	NR
SatI	G
ScaI	ScaI	NR	NR
SchI	Tango™		NR
SdaI	SdaI	NR
SduI	SduI	NR	NR
SfaAI	Tango™		NR
SfiI (50°C)	G		NR
SgsI	Tango
SmaI (30°C)	Tango™		NR
SmiI (30°C)	O	NR
SmoI (55°C)	Tango™
SmuI	Tango™
SsiI	O	NR
SspI	G
TaaI (65°C)	Tango™
TaiI (65°C)	R
TaqI (65°C)	TaqI
TasI (65°C)	B		NR
TatI (65°C)	Tango™	*	NR
TauI (55°C)	B		NR
Tru1I (65°C)	R
TscAI (65°C)	Tango™
TsoI (55°C)	G +SAM	+SAM	+SAM
TstI	R	NR
Van91I	R
VspI	O
XagI	R
XapI	Tango™
XbaI	Tango™
XceI	Tango™		NR
XhoI	R
XmaJI	Tango™
XmiI	B
Nb.Bpu10I	R
Nt.Bpu10I	R	NR
I-SceI	Tango™

Note
Yellow shaded buffers yield 50-100% and grey shaded buffers yield 20-50% of restriction enzyme activity.
NR: Buffer is not recommended, because of high star activity.
* Star activity appears at 5-fold overdigestion (5 units x 1 hour).
Optimal temperature of enzymes enlisted is 37°C, unless otherwise indicated in parenthesis.

catalog@fermentas.com

Updated sausio 24, 2008 16:02