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AN INNOVATION FROM FERMENTAS: FastDigest® Restriction Enzymes

Save Time - Digest DNA in Just 5 min!

Digestion of DNA with restriction enzymes is a time consuming step in cloning and clone analysis. DNA digestions typically last for one hour, and often DNA is incubated with the enzymes overnight. The PureExtreme® Quality of our enzymes has enabled us to develop a new line of products - the Fermentas FastDigest® Restriction Enzymes, which are specifically formulated to cleave DNA in just 5 minutes.
High quality DNA is crucial for efficient DNA digestion in 5 minutes. We recommend the Fermentas GeneJET™ Plasmid Miniprep Kit to purify DNA for FastDigest®. While FastDigest® is compatible with DNA purified using kits from other suppliers, Fermentas guarantees the performance of our products, which are tested under the most rigorous and demanding conditions.

Features

• Single, double and triple digestions of plasmid DNA in just 5 minutes.
• Enhanced performance in one-hour DNA cleavage reactions.
• A single reaction buffer for all FastDigest® enzymes.
• FastDigest® buffer is compatible with downstream applications.

Applications

• Fast clone analysis.
• Fast preparation of vectors for cloning.
• Fast digestion of PCR products.
• Fast digestion of genomic DNA.
• Standard DNA cleavage reactions.

Note

• Low quality plasmid DNA may require longer incubation times.
• Maximal ligation efficiency may be achieved using gel-purified DNA.

Clone analysis: five minute digestion with FastDigest® enzymes. A recombinant pJET1 plasmid containing 953 bp insert was purified from recombinant E.coli culture using the GeneJET™ Plasmid Miniprep Kit and digested in 5 minutes with FastDigest® enzymes. M - GeneRuler™ Express DNA Ladder 1 - plasmid containing insert 2 - plasmid containing insert, digested with FastDigest® NotI 3 - plasmid containing insert, digested with FastDigest® BamHI 4 - plasmid containing insert, double digested with FastDigest® NotI and FastDigest® BamHI 5 - plasmid containing insert in the opposite orientation, double digested with FastDigest® NotI and FastDigest® BamHI

We offer the following FastDigest® enzymes: 

Enzyme	Prototype	Specificity 5'=>3'	Catalog #	Note	Single letter code: R = G or A Y = C or T W = A or T M = A or C K = G or T S = C or G H = A, C or T V = A, C or G B = C, G or T D = A, G or T N = G, A, T or C
FastDigest® AccI (XmiI)	AccI	GT^MKAC	FD1484	not recommended for fast digestion of PCR products
FastDigest® Acc65I	KpnI (GGTAC^C)	G^GTACC	FD0904
FastDigest® AciI (SsiI)	AciI	CCGC(-3/-1)^	FD1794
FastDigest® AclI (Psp1406I)	AclI	AA^CGTT	FD0944
FastDigest® AfeI (Eco47III)	Eco47III	AGC^GCT	FD0324
FastDigest® AflII (BspTI) new	AflII	C^TTAAG	FD0834
FastDigest® AgeI (BshTI)	AgeI	A^CCGGT	FD1464
FastDigest® AjuI new	AjuI	^(7/12)GAA(N)7TTGG(11/6)^	FD1954
FastDigest® AleI (OliI)	OliI	CACNN^NNGTG	FD1634
FastDigest® Alw21I	HgiAI	GWGCW^C	FD0024
FastDigest® Alw26I	BsmAI	GTCTC(1/5)^	FD0034
FastDigest® AlwNI (CaiI)	AlwNI	CAGNNN^CTG	FD1394
FastDigest® ApaI	ApaI	GGGCC^C	FD1414
FastDigest® ApaLI (Alw44I)	ApaLI	G^TGCAC	FD0044
FastDigest® AscI (SgsI)	AscI	GG^CGCGCC	FD1894
FastDigest® AseI (VspI)	VspI	AT^TAAT	FD0914
FastDigest® AsiSI (SfaAI)	SgfI	GCGAT^CGC	FD2094
FastDigest® AvaI (Eco88I)	AvaI	C^YCGRG	FD0384
FastDigest® AvaII (Eco47I)	AvaII	G^GWCC	FD0314
FastDigest® AvrII (XmaJI)	AvrII	C^CTAGG	FD1564
FastDigest® BamHI	BamHI	G^GATCC	FD0054
FastDigest® BanI (BshNI)	HgiCI	G^GYRCC	FD1004
FastDigest® BbsI (BpiI)	BbvII	GAAGAC (2/6)^	FD1014
FastDigest® BbvI (Lsp1109I)	BbvI	GCAGC(8/12)^	FD2074
FastDigest® BclI	BclI	T^GATCA	FD0724
FastDigest® BfaI (FspBI)	MaeI	C^TAG	FD1764
FastDigest® BglI	BglI	GCCNNNN^NGGC	FD0074
FastDigest® BglII	BglII	A^GATCT	FD0083 FD0084
FastDigest® BlpI (Bpu1102I)	EspI	GC^TNAGC	FD0094
FastDigest® Bme1580I (BseSI)	BseSI	GKGCM^C	FD1444
FastDigest® BmtI (BspOI)	NheI (G^CTAGC)	GCTAG^C	FD2044
FastDigest® BsaAI (Ppu21I) new	BsaAI	YAC^GTR	FD1974
FastDigest® BsaHI (Hin1I)	AcyI	GR^CGYC	FD0474
FastDigest® BsaJI (BseDI)	SecI	C^CNNGG	FD1084
FastDigest® BseGI	FokI (GGATG(9/13)^)	GGATGNN^	FD0874
FastDigest® BseNI	BsrI	ACTGG(1/-1)^	FD0884
FastDigest® Bsh1236I	FnuDII	CG^CG	FD0924
FastDigest® BslI (BseLI)	BsiYI	CCNNNNN^NNGG	FD1204
FastDigest® BsmBI (Esp3I) new	Esp3I	CGTCTC(1/5)^	FD0454
FastDigest® Bsp119I	AsuII	TT^CGAA	FD0124
FastDigest® Bsp120I	ApaI (GGGCC^C)	G^GGCCC	FD0134
FastDigest® Bsp1286I (SduI) new	SduI	GDGCH^C	FD0654
FastDigest® Bsp1407I	Bsp1407I	T^GTACA	FD0933 FD0934
FastDigest® BspHI (PagI) new	BspHI	T^CATGA	FD1284
FastDigest® BsrBI (MbiI)	BsrBI	CCGCTC(-3/-3)^	FD1274
FastDigest® BsrFI (Cfr10I)	Cfr10I	R^CCGGY	FD0184
FastDigest® Bsu36I (Eco81I) new	SauI	CC^TNAGG	FD0374
FastDigest® BstXI	BstXI	CCANNNNN^NTGG	FD1024
FastDigest® BstZ17I (Bst1107I)	SnaI	GTA^TAC	FD0704
FastDigest® ClaI (Bsu15I)	ClaI	AT^CGAT	FD0143 FD0144
FastDigest® Csp6I	RsaI (GT^AC)	G^TAC	FD0214
FastDigest® DdeI (HpyF3I)	DdeI	C^TNAG	FD1884
FastDigest® DpnI	DpnI	Gm6A^TC	FD1703 FD1704	not applicable for fast digestion of PCR products
FastDigest® DraI	AhaIII	TTT^AAA	FD0224
FastDigest® DraIII (AdeI)	DraIII	CACNNN^GTG	FD1234
FastDigest® DrdI (AasI) new	DrdI	GACNNNN^NNGTC	FD1724
FastDigest® EagI (Eco52I)	XmaIII	C^GGCCG	FD0334
FastDigest® Eam1105I	Eam1105I	GACNNN^NNGTC	FD0244
FastDigest® EarI (Eam1104I) new	Ksp632I	CTCTTC(1/4)^	FD0234
FastDigest® Ecl136II	SacI (GAGCT^C)	GAG^CTC	FD0254
FastDigest® Eco31I	Eco31I	GGTCTC(1/5)^	FD0293 FD0294
FastDigest® Eco91I	BstEII	G^GTNACC	FD0394
FastDigest® EcoNI (XagI)	EcoNI	CCTNN^NNNAGG	FD1304
FastDigest® EcoO109I	DraII	RG^GNCCY	FD0264
FastDigest® EcoRI	EcoRI	G^AATTC	FD0274
FastDigest® EcoRV (Eco32I)	EcoRV	GAT^ATC	FD0303 FD0304
FastDigest® EheI	NarI (GG^CGCC)	GGC^GCC	FD0443 FD0444
FastDigest® Fnu4HI (SatI)	Fnu4HI	GC^NGC	FD1644
FastDigest® FspI (NsbI)	MstI	TGC^GCA	FD1224
FastDigest® FspAI	FspAI	RTGC^GCAY	FD1664
FastDigest® HaeIII (BsuRI)	HaeIII	GG^CC	FD0154
FastDigest® HhaI	HhaI	GCG^C	FD1854
FastDigest® HincII	HindII	GTY^RAC	FD0494
FastDigest® HindIII	HindIII	A^AGCTT	FD0504
FastDigest® HinfI	HinfI	G^ANTC	FD0804
FastDigest® HinP1I (Hin6I)	HhaI (GCG^C)	G^CGC	FD0484
FastDigest® HpaI (KspAI)	HpaI	GTT^AAC	FD1034
FastDigest® HpaII	HpaII	C^CGG	FD0514
FastDigest® Hpy8I	MjaIV	GTN^NAC	FD1574
FastDigest® HpyF10VI	MwoI	GCNNNNN^NNGC	FD1734
FastDigest® Kpn2I	BspMI	T^CCGGA	FD0534
FastDigest® KpnI	KpnI	GGTAC^C	FD0524
FastDigest® MboI	MboI	^GATC	FD0814
FastDigest® MboII	MboII	GAAGA(8/7)^	FD0824
FastDigest® MfeI (MunI)	MfeI	C^AATTG	FD0753 FD0754
FastDigest® MluI	MluI	A^CGCGT	FD0564
FastDigest® MlyI (SchI)	PleI (GAGTC(4/5)^)	GAGTC(5/5)^	FD1374
FastDigest® MnlI	MnlI	CCTC(7/6)^	FD1074
FastDigest® MreI	Sse232I	CG^CCGGCG	FD2024
FastDigest® MscI (MlsI)	BalI	TGG^CCA	FD1214
FastDigest® MslI (RseI)	MslI	CAYNN^NNRTG	FD2004
FastDigest® MspI	HpaII	C^CGG	FD0544
FastDigest® MssI	PmeI	GTTT^AAAC	FD1344
FastDigest® MvaI	EcoRII (^CCWGG)	CC^WGG	FD0554
FastDigest® Mva1269I	BsmI	GAATGC(1/-1)^	FD0964
FastDigest® NaeI (PdiI) new	NaeI	GCC^GGC	FD1524
FastDigest® NciI (BcnI)	CauII	CC^SGG	FD0064
FastDigest® NcoI	NcoI	C^CATGG	FD0573 FD0574
FastDigest® NdeI	NdeI	CA^TATG	FD0583 FD0584	not recommended for fast digestion of PCR products
FastDigest® NheI	NheI	G^CTAGC	FD0973 FD0974
FastDigest® NlaIII (Hin1II) new	NlaIII	CATG^	FD1834
FastDigest® NlaIV (BspLI)	NlaIV	GGN^NCC	FD1154
FastDigest® NmuCI	Tsp45I	^GTSAC	FD1514
FastDigest® NotI	NotI	GC^GGCCGC	FD0593 FD0594
FastDigest® NsiI (Mph1103I)	AvaIII	ATGCA^T	FD0734
FastDigest® NspI (XceI)	NspI	RCATG^Y	FD1474
FastDigest® PdmI	XmnI	GAANN^NNTTC	FD1534
FastDigest® PflMI (Van91I)	PflMI	CCANNNN^NTGG	FD0714
FastDigest® PfoI	PfoI	T^CCNGGA	FD1754
FastDigest® PmlI (Eco72I)	PmaCI	CAC^GTG	FD0364
FastDigest® PpuMI (Psp5II)	PpuMI	RG^GWCCY	FD0764
FastDigest® PshAI (BoxI)	PshAI	GACNN^NNGTC	FD1434
FastDigest® PsiI (AanI)	PsiI	TTA^TAA	FD2064
FastDigest® PstI	PstI	CTGCA^G	FD0614
FastDigest® PsuI	XhoII	R^GATCY	FD1554
FastDigest® PsyI	Tth111I	GACN^NNGTC	FD1334
FastDigest® PvuI	PvuI	CGAT^CG	FD0624
FastDigest® PvuII	PvuII	CAG^CTG	FD0634
FastDigest® RsaI	RsaI	GT^AC	FD1124
FastDigest® SacI	SacI	GAGCT^C	FD1133 FD1134
FastDigest® SalI	SalI	G^TCGAC	FD0644	not recommended for fast digestion of PCR products
FastDigest® SapI (LguI)	SapI	GCTCTTC(1/4)^	FD1934
FastDigest® Sau96I (Cfr13I)	AsuI	G^GNCC	FD0194
FastDigest® SbfI (SdaI)	Sse8387I	CCTGCA^GG	FD1194
FastDigest® ScaI	ScaI	AGT^ACT	FD0434
FastDigest® ScrFI (Bme1390I)	ScrFI	CC^NGG	FD1424
FastDigest® SfcI (BfmI)	SfeI	C^TRYAG	FD1164
FastDigest® SmaI	SmaI	CCC^GGG	FD0663 FD0664
FastDigest® SnaBI (Eco105I)	SnaBI	TAC^GTA	FD0404
FastDigest® SpeI (BcuI)	SpeI	A^CTAGT	FD1253 FD1254
FastDigest® SphI (PaeI)	SphI	GCATG^C	FD0604
FastDigest® SspI	SspI	AAT^ATT	FD0774
FastDigest® StyI (Eco130I)	StyI	C^CWWGG	FD0414
FastDigest® StuI (Eco147I)	StuI	AGG^CCT	FD0424
FastDigest® SwaI (SmiI)	SwaI	ATTT^AAAT	FD1244
FastDigest® TaaI	Tsp4CI	ACN^GT	FD1364
FastDigest® TaiI	MaeII (A^CGT)	ACGT^	FD1144
FastDigest® TaqI	TaqI	T^CGA	FD0674
FastDigest® Tru1I	MseI	T^TAA	FD0984
FastDigest® Tsp509I (TasI)	TspEI	^AATT	FD1354
FastDigest® TspRI (TscAI)	TspRI	CASTG^(2/-7)^	FD2104
FastDigest® XapI	ApoI	R^AATTY	FD1383 FD1384
FastDigest® XbaI	XbaI	T^CTAGA	FD0684
FastDigest® XhoI	XhoI	C^TCGAG	FD0694

 

Analyze Your Clones 3 Times Faster

Step	Conventional protocol	Fast protocol	Fermentas products for the fast protocol
Miniprep purification and DNA pipetting	40 min	15 min	GeneJET™ Plasmid Miniprep Kit
Preparation of digestion Master mix	6 min	6 min	FastDigest® restriction enzymes (see the table above)
Digestion of DNA	60 min	5 min
Electrophoresis	40 min	17 min	ZipRuler™ Express DNA Ladder Set
			GeneRuler™ Express DNA Ladders
			FastRuler™ Middle Range DNA Ladder
Total time:	146 min	43 min

I. Digestion of single sample

1. Purify DNA from 1.5 ml of E.coli culture using GeneJET™ Plasmid Miniprep Kit.
2. Prepare the following reaction mixture in a thin-wall tube at room temperature:

   Water, nuclease-free	15 µl
   10X FastDigest® buffer	2 µl
   Plasmid DNA (up to 1 µg)	2 µl
   FastDigest® enzyme	1 µl

3. Mix gently and spin down.
4. Incubate* at 37°C for 5 minutes to digest DNA.
   * Use a heat block or a water thermostat. Air thermostats are not recommended due to slow heat transfer to the reaction mixture.
5. Add 4 µl of 6X DNA Loading Dye into each tube and mix.
6. Load on a 0.8-1% agarose gel and run electrophoresis for 10-20 min. Use a DNA ladder from the table above.

II. Digestion of multiple samples

1. Purify DNA from 1.5 ml of E.coli culture using GeneJET™ Plasmid Miniprep Kit.
2. Pipette 2 µl (up to 1 µg) aliquots of miniprep DNA into thin-wall tubes.
3. Prepare the following master mix at room temperature:

   Water, nuclease-free	(number of samples + 1) x 15 µl
   10X FastDigest® buffer	(number of samples + 1) x 2 µl
   FastDigest® enzyme	(number of samples + 1) x 1 µl

4. Add 18 µl of the master mix into each tube with plasmid DNA.
5. Mix gently and spin down.
6. Incubate* at 37°C for 5 minutes to digest DNA.
   * Use a heat block or a water thermostat. Air thermostats are not recommended due to slow heat transfer to the reaction mixture.
7. Add 4 µl of 6X DNA Loading Dye into each tube and mix.
8. Load on a 0.8-1% agarose gel and run electrophoresis for 10-20 min. Use a DNA ladder from the table above.

1. Prepare the following reaction mixture in a thin-wall tube at room temperature:
   

   Water, nuclease-free	variable
   10X FastDigest® buffer	2 µl
   Plasmid DNA (up to 1 µg)**	variable
   FastDigest® enzyme	1 µl
   Final volume	20 µl

2. Mix gently and spin down.
3. Incubate* at 37°C for 5 minutes to digest DNA.
   * Use a heat block or a water thermostat. Air thermostats are not recommended due to slow heat transfer to the reaction mixture.
   ** For digestion of larger amounts of DNA, scale-up the reaction mixture:

   DNA**	1 µg	2 µg	3 µg	4 µg	5 µg
   FastDigest® enzyme	1 µl	2 µl	3 µl	4 µl	5 µl
   10X FastDigest® buffer	2 µl	2 µl	3 µl	4 µl	5 µl
   Final volume	20 µl	20 µl	30 µl	40 µl	50 µl

1. Prepare the following reaction mixture in a thin-wall tube at room temperature:
   

   Water, nuclease-free	variable
   10X FastDigest® buffer	5 µl
   Genomic DNA (up to 5 µg)*	variable (up to 25 µl)
   FastDigest® enzyme	1 µl or 5 µl
   Final volume	50 µl

2. Mix gently and spin down.
3. Incubate** as indicated in the enzyme description or Certificate of Analysis of each enzyme.
   * For digestion of larger amounts of DNA, scale-up the reaction mixture. However, reaction volumes higher than 50 µl may require longer incubation time due to slower heat transfer.
   ** Use a heat block or a water thermostat. Air thermostats are not recommended due to slow heat transfer to the reaction mixture.

I. Digestion of single sample

1. Perform PCR**.
2. Prepare the following reaction mixture in a thin-wall tube at room temperature:
   

   Water, nuclease-free	17 µl
   10X FastDigest® buffer	2 µl
   PCR product (~0.2 µg)	10 µl
   FastDigest® enzyme	1 µl

3. Mix gently and spin down.
4. Incubate* at 37°C for 5 minutes to digest DNA.
   * Use a heat block or a water thermostat. Air thermostats are not recommended due to slow heat transfer to the reaction mixture.

II. Digestion of multiple samples

1. Pipette 10 µl (~0.2 µg) of each PCR product into thin-wall tubes.
2. Prepare the following master mix at room temperature:
   

   Water, nuclease-free	(number of samples+1) x 17 µl
   10X FastDigest® buffer	(number of samples+1) x 2 µl
   FastDigest® enzyme	(number of samples+1) x 1 µl

3. Add 20 µl of the master mix into each tube.
4. Mix gently and spin down.
5. Incubate* at 37°C for 5 minutes to digest DNA.
   * Use a heat block or a water thermostat. Air thermostats are not recommended due to slow heat transfer to the reaction mixture.

Note

• PCR additives such as DMSO or glycerol may affect the cleavage efficiency of FastDigest® enzymes or cause star activity.
• In some cases, the context of the target sequence may affect cleavage of PCR products, resulting in incomplete cleavage after 5 minutes. In these cases, a 30 min incubation with the FastDigest® enzyme is recommended.
• When introducing restriction enzyme sites into primers for subsequent digestion and cloning of a PCR product, refer to the Table “Cleavage efficiency close to the termini of PCR fragments” to define the number of extra bases required for efficient cleavage.
• For double digestions of PCR products reduce the amount of water by 1 µl and add 1 µl of the second FastDigest® enzyme to the reaction mixture or master mix.
• For cloning applications, purification of PCR products prior to digestion is highly recommended to remove the active thermophilic DNA polymerase still present in the reaction mixture. DNA polymerases may alter the ends of the cleaved DNA and reduce the ligation yield.

I. Digestion of single sample

1. Prepare the following reaction mixture in a thin-wall tube at room temperature:
   

   Water, nuclease-free	variable
   10X FastDigest® buffer	2 µl
   DNA (up to 1 µg)	variable
   FastDigest® enzyme 1	1 µl
   FastDigest® enzyme 2	1 µl
   Final volume	20 µl

2. Mix gently and spin down.
3. Incubate* at 37°C for 5 minutes to digest DNA.
   * Use a heat block or a water thermostat. Air thermostats are not recommended due to slow heat transfer to the reaction mixture.

II. Digestion of multiple samples

1. Pipette 2 µl (up to 1 µg) aliquots of DNA into thin-wall tubes at room temperature.
2. Prepare the following master mix at room temperature:



Water, nuclease-free	(number of samples + 1) x 14 µl
10X FastDigest® buffer	(number of samples + 1) x 2 µl
FastDigest® enzyme 1	(number of samples + 1) x 1 µl
FastDigest® enzyme 2	(number of samples + 1) x 1 µl

Note
If your substrate DNA is in a smaller volume, increase the volume of water, accordingly.

3. Mix gently and spin down.
4. Incubate* at 37°C for 5 minutes to digest DNA.
   * Use a heat block or a water thermostat. Air thermostats are not recommended due to slow heat transfer to the reaction mixture.

Note

• Low quality plasmid DNA may require longer incubation time.
• FastDigest® enzymes are suitable for any one-hour digestion reactions. They generally result in more efficient cleavage of difficult-to-cleave targets.

1. Prepare the following reaction mixture in a thin-wall tube at room temperature:
   

   Water, nuclease-free	variable
   10X FastDigest® buffer	2 µl
   DNA (up to 1 µg)	variable
   FastDigest® 1	1 µl
   FastDigest® 2	1 µl
   FastDigest® 3	1 µl
   Final volume	20 µl

2. Mix gently and spin down.
3. Incubate* at 37°C for 5 minutes to digest DNA.
   * Use a heat block or a water thermostat. Air thermostats are not recommended due to slow heat transfer to the reaction mixture.

Updated gegužės 07, 2008 13:47