FastDigest Restriction Enzymes—Thermo Scientific

One buffer for 176 enzymes. It's that easy.

Thermo Scientific FastDigest restriction enzymes are an advanced line of enzymes which offer:

Complete digestion in 5-15 minutes
100% buffer compatibility with downstream applications
Direct loading on gels

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Supported by over 40 years of experience

Through the FastDigest collection, you have access to one of the largest collections of restriction enzymes in the industry. Decades of expertise in enzymology, the large selection of enzyme isoschizomers, and the ability to produce enzymes of exceptional purity from our state-of-the-art manufacturing facilities helped enable the creation of the first simplified restriction enzyme system using a universal buffer.

Advantages of FastDigest restriction enzymes

	FastDigest restriction enzymes	Conventional restriction enzymes
Buffer system	One Universal Buffer	Up to 20 Buffers
Double/multiple restriction digestion	Unlimited – all 176 enzymes are 100% active in one buffer	Limited by buffer compatibility
Reaction Time	5–15 minutes	1 hour–overnight
Direct loading on gels	Yes	No
Downstream applications	100% buffer compatibility	Partial compatibility
Star activity	No	Yes
Activity definintion	1 µL of FastDigest enzyme cleaves1 µg of substrate DNA in 5 to 15 minutes in FastDigest buffer	1 unit of enzyme hydrolyzes 1µg of lambda DNA in 60 minutes in an optimal buffer for an enzyme

100% activity in a single buffer

The FastDigest Green Buffer and Thermo Scientific FastDigest Buffer are proprietary digestion buffers which support 100% activity of all FastDigest restriction enzymes. This system allows for double and multiple digestions with any combination of enzymes. No sequential digestions or buffer changes are needed.

Five minute single, double and triple digestions in FastDigest Green Buffer

Five minute plasmid DNA digestions with:

Lane M: Thermo Scientific GeneRuler Express Ladder (Cat. No. SM1553)
Lane 1: Control, undigested plasmid DNA
Lane 2: FastDigest EcoRI
Lane 3: FastDigest EcoRI and FastDigest KpnI
Lane 4: FastDigest EcoRI, FastDigest KpnI and FastDigest SmaI

100% buffer compatibility with downstream applications

DNA/RNA modifying enzymes, such as ligases, phosphatases, kinases and mesophilic DNA polymerases have 100% activity in FastDigest and FastDigest Green Buffer. Therefore, enzymes used in downstream applications can be directly added to the FastDigest reaction mix. No buffer changes or purification steps are needed.

DNA/RNA Modifying Enzyme	Activity in FastDigest Green Buffer/FastDigest Buffer
DNA Polymerase I, E. coli	100%
Klenow Fragment	100%
Klenow Fragment, exo-	100%
T4 DNA Polymerase	100%
T7 DNA Polymerase	100%
T4 DNA Ligase*	75–100%
FastAP Thermosensitive Alkaline Phosphatase	100%
T4 Polynucleotide Kinase	100%

* 0.5 mM ATP is required for T4 DNA Ligase activity.

Complete digestion in 5-15 minutes

Double and multiple digestions in one buffer in 5-15 minutes:

Saves time and effort, increasing throughput.
Digestion times are provided for all types of DNA templates (plasmid DNA, PCR product, genomic DNA).
No overnight digestions are required for any template.
Star activity is eliminated due to short reaction times.

Conventional restriction enzymes Sequential digestion		FastDigest restriction enzymes One reaction mixture
Reaction setup for Apal	~2 min	Reaction set up with FastDigest ApaI and XhoI	~2 min
Incubation	60 min	Incubation	5 min
Reaction setup for Xhol	~2 min	—	—
Incubation	60 min	—	—
TOTAL TIME	>2 hours	TOTAL TIME	7 min

Direct loading on gels

As an added convenience we developed the FastDigest Green Buffer which offers the same performance as the colorless FastDigest Buffer but enables direct loading of the reaction mixture on gels. The 10X FastDigest Green Buffer includes a density reagent and two tracking dyes for direct loading. The blue dye migrates with 3–5 kb DNA fragments in a 1% agarose gel and has an excitation peak of 424 nm. The yellow dye migrates faster than 10 bp DNA fragments in a 1% agarose gel and has an excitation peak of 615 nm.

FastDigest Green Buffer

Reaction mixture containing FastDigest Green Buffer:

Lane 1: Loaded into gel well, before electrophoresis
Lane 2: Separated, after electrophoresis

Short protocol times with no star activity

FastDigest enzymes in combination with FastDigest (Green) Buffer are designed to eliminate star activity:

Short incubation time (5–15 min), no need for prolonged digestions.
Universal buffer with optimal pH and salt concentration for all enzymes.
Optimal enzyme and glycerol concentrations.

Conventional restriction enzymes may display star or "relaxed" activity due to prolonged incubation times, high enzyme and/or glycerol concentration, high pH values or low ionic strength. By addressing all these issues, FastDigest enzymes enable single, double and even triple digestion in 5 minutes without any signs of star activity.

Five minute single and double digestion of plasmid DNA with no signs of star activity

Five minute plasmid DNA digestions with:

Lane 1: FastDigest SmaI
Lane 2: FastDigest EcoRI
Lane 3: FastDigest SmaI and FastDigest EcoRI double digest
Lane M: GeneRuler 1 kb Plus DNA Ladder (Cat. No. SM1333)
Lane C: Control, Undigested plasmid DNA

References

Applications	Publications
FastDigest EcoRI and BamHI restriction enzymes were used to prepare vector for Golden Gate cloning method.	Wang T, Wang D, Lyu Y, et al. (2018) Construction of a high-efficiency cloning system using the Golden Gate method and I-SceI endonuclease for targeted gene replacement in Bacillus anthracis. J Biotechnol 271:8–16.
FastDigest BpiI (BbsI) Type IIS restriction enzyme was used to construct gRNA.	Kurata M, Wolf NK, Lahr WS, et al. (2018) Highly multiplexed genome engineering using CRISPR/Cas9 gRNA arrays. PLoS ONE 13(9):e0198714.
FastDigest TaaI restriction enzyme was used in RFLP analysis of PCR products.	Fong WY, Ho CC, Poon WT. (2017) Comparison of Direct Sequencing, Real-Time PCR-High Resolution Melt (PCR-HRM) and PCR-Restriction Fragment Length Polymorphism (PCR-RFLP) Analysis for Genotyping of Common Thiopurine Intolerant Variant Alleles NUDT15 c.415C>T and TPMT c.719A>G (TPMT3C). Diagnostics (Basel)* 7(2):27.
Fragmentation of genomic DNA with FastDigest HindIII restriction enzymes prior to ddPCR.	Li B, Kaushik S, Kalinowski P, et al. (2018) Droplet digital PCR shows the D-Loop to be an error prone locus for mitochondrial DNA copy number determination. Sci Rep 8(1):11392.