Thermo Scientific FastDigest Restriction Enzymes are an advanced line of enzymes offering fast and complete digestion of DNA in a single universal buffer. With over 30 years experience in support of restriction enzyme research, our FastDigest enzymes are a top customer pick offering 176 unique specificities in formulations that support direct loading of reaction mixtures on gels.
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Over the course of 30 years, our search for restriction enzymes led us to acquire one of the largest collections of restriction enzyme-producing bacterial strains in the industry. Our wide selection of isoschizomers and ability to produce enzymes of exceptional purity enabled us to create the unique system of 176 FastDigest restriction enzymes formulated to have 100% activity in a single buffer.
| Conventional restriction enzymes | FastDigest restriction enzymes | |
|---|---|---|
| Buffer system | Up to 20 Buffers | One Universal Buffer |
| Double/multiple restriction digestion | Limited by buffer compatibility | Any enzyme combination possible |
| Reaction Time | 1 hour–overnight | 5–15 minutes |
| Direct loading on gels | No |
Yes |
| Downstream applications | Partial compatibility |
100% buffer compatibility |
| Star activity | Yes | No |
| Activity definintion | 1 unit of enzyme hydrolyzes 1µg of lambda DNA in 60 minutes in an optimal buffer for an enzyme | 1 µL of FastDigest enzyme cleaves1 µg of substrate DNA in 5 to 15 minutes in FastDigest 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 plasmid DNA digestions with:
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.
Double and multiple digestions in one buffer in 5-15 minutes:
| 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.
Reaction mixture containing FastDigest Green Buffer:
FastDigest enzymes in combination with FastDigest (Green) Buffer are designed to eliminate star activity:
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 plasmid DNA digestions with:
For Research Use Only. Not for use in diagnostic procedures.
