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 |
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
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