Thermo Scientific FastDigest restriction enzymes are an advanced line of enzymes which offer:
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.
|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|
|Activity definition||1 µL of FastDigest enzyme cleaves 1 µg of substrate DNA in 5 to 15 minutes in FastDigest buffer||1 unit of enzyme hydrolyzes 1 µg of lambda DNA in 60 minutes in an optimal buffer for an enzyme|
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||Cat. No.||Activity in FastDigest Green Buffer/FastDigest Buffer|
|DNA Polymerase I, E. coli||EP0041|
|Klenow Fragment, exo-||EP0421|
|T4 DNA Polymerase||EP0061|
|T7 DNA Polymerase||EP0081||100%|
|T4 DNA Ligase*||EL0011|
|FastAP Thermosensitive Alkaline Phosphatase||EF0651|
|T4 Polynucleotide Kinase||EK0031|
* 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|
|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||—||—|
|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:
|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 (TPMT*3C). 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.|
For Research Use Only. Not for use in diagnostic procedures.