Description

Thermo Scientific FastDigest enzymes are an advanced

line of restriction enzymes for rapid DNA digestion. All
FastDigest™ enzymes are 100% active in the universal
PRODUCT INFORMATION FastDigest and FastDigest Green buffers and are able to
digest DNA in 5-15 minutes. This enables any combination of
Thermo Scientific restriction enzymes to work simultaneously in one reaction
FastDigest HinfI tube and eliminates the need for sequential digestions.
FastDigest enzymes can be used to digest plasmid, genomic
and viral DNA as well as PCR products and do not show star
#FD0804 400 µL (for 400 rxns) activity even in prolonged incubations.
Enzymes used in common downstream applications such as
Lot: ________ Expiry Date: _______
ligation, blunting and dephosphorylation reactions also have
5'... G↓A N T C...3' 100% activity in FastDigest and FastDigest Green Buffer.
3'... C T N A ↑ G ...5'
FastDigest Green Buffer includes a density reagent along
with blue and yellow tracking dyes that allow for direct
Supplied with: 1 mL of 10X FastDigest Buffer loading of the reaction mixtures on a gel.
1 mL of 10X FastDigest Green Buffer The blue dye of the FastDigest Green Buffer migrates with
3-5 kb DNA fragments in a 1% agarose gel and has an
Store at -20°C
excitation peak at 424 nm.
The yellow dye of the FastDigest Green Buffer migrates
faster than 10 bp DNA fragments in a 1% agarose gel and
has an excitation peak at 615 nm.
For applications that require analysis by fluorescence
excitation FastDigest Buffer is recommended, as the dyes of
the FastDigest Green Buffer may interfere with some
BSA included fluorescence measurements.
www.thermoscientific.com/onebio
Rev.10 V
Recommended Reaction Conditions CERTIFICATE OF ANALYSIS
• 1X FastDigest Buffer or 1X FastDigest Green Buffer. Functional Activity Test
• Incubation at 37°C. 1 µg of lambda DNA was completely digested with 1 µL
• 1 µL of FastDigest HinfI is formulated to digest up to: of the enzyme in 5 minutes at 37°C in 20 µL of reaction
– 1 µg of lambda DNA in 5 min. mixture.
– 1 µg of plasmid DNA in 5 min. Ligation and Recleavage (L/R) Assay
– 0.2 µg of PCR product in 5 min. The ligation and recleavage assay was replaced with LO
– 1 µg of genomic DNA in 5 min, or 5 µg of genomic test after validating experiments showed LO test ability to
DNA in 30 min. trace nuclease and phosphatase activities with sensitivity
Thermal Inactivation: Incubation at 65°C for 20 min. that is higher than L/R by a factor of 100.
Methylation Effects on Digestion Labeled Oligonucleotide (LO) Assay
Dam: never overlaps – no effect. No detectable degradation of single-stranded or double-
Dcm: never overlaps – no effect. stranded oligonucleotides occured during incubation with
CpG: may overlap – cleavage impaired. 1 µL of FastDigest HinfI for 1 hour.
EcoKI: never overlaps – no effect. Prolonged Incubation / Star Activity Assay
EcoBI: may overlap – blocked. No detectable degradation of 1 µg of lambda DNA due to
Compatible ends nuclease contamination or star activity occurred during
Check www.thermoscientific.com/research for the list of incubation with 1µLl of FastDigest HinfI for 16 hours.
restriction enzymes producing compatible ends. Quality authorized by: Jurgita Zilinskiene
Number of Recognition Sites in DNA
λ ΦX174 pBR322 pUC57 pUC18/19 pTZ19R/U M13mp18/19
148 21 10 5 6 9 27

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Protocol for Fast Digestion of Different DNA
1. Combine the following reaction components at room temperature in the order indicated:
Plasmid DNA PCR product Genomic DNA
Water, nuclease-free (#R0581) 15 µL 17 µL 30 µL
10X FastDigest or 10X FastDigest Green Buffer 2 µL 2 µL 5 µL
DNA 2 µL (up to 1 µg) 10 µL (~0.2 µg) 10 µL (5 µg)
FastDigest enzyme 1 µL 1 µL 5 µL
Total volume: 20 µL 30 µL 50 µL
2. Mix gently and spin down.
3. Incubate at 37°C in a heat block or water thermostat for 5 min.
Optional: Inactivate the enzyme by heating for 20 min at 65°C.
4. If the FastDigest Green Buffer was used in the reaction, load an aliquot of the reaction mixture directly on a gel.
Note: The FastDigest Green Buffer can be used as an electrophoresis loading buffer for any DNA sample at a final 1X concentration.
Higher concentrations of FastDigest Green Buffer in the sample supply excess salt concentration which may alter DNA mobility.
Double and Multiple Digestion of DNA
• The combined volume of the enzymes in the reaction mixture should not exceed 1/10 of the total reaction volume.
• Use 1 µL of each enzyme and scale up the reaction conditions appropriately.
• If the enzymes require different reaction temperatures, start with the enzyme that requires a lower temperature, then add the
second enzyme and incubate at the higher temperature.
Scaling up Plasmid DNA Digestion Reaction
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 or 10X FastDigest Green Buffer 2 µL 2 µL 3 µL 4 µL 5 µL
Total volume: 20 µL 20 µL 30 µL 40 µL 50 µL
Note: Increase the incubation time by 3-5 min if the total reaction volume exceeds 20 µL. Use water thermostat, air thermostats are not
recommended due to the slow transfer of heat to the reaction mixture.
Recommendations for PCR product digestion
• When introducing restriction enzyme sites into primers for subsequent digestion and cloning of a PCR product, refer to
www.thermoscientific.com/fd, Reaction Conditions Guide, to define the number of extra bases required for efficient cleavage.
• Use Thermo Scientific GeneJET PCR Purification Kit, #K0701 to purify PCR product prior digestion in following cases:
− When PCR additives such as DMSO or glycerol where used, as they may affect the cleavage efficiency or cause star
activity.
− When PCR Product will be used for cloning. Active thermophilic DNA polymerase still present in PCR mixture may alter the
ends of the cleaved DNA and reduce the ligation efficiency.
Activity of DNA Modifying Enzymes in FastDigest and FastDigest Green Buffers, %
Thermo Scientific FastAP Thermosensitive Alkaline Phosphatase, #EF0651 100
T4 DNA Ligase*, #EL0014 75-100
Klenow Fragment, #EP0051 100
T4 DNA Polymerase, #EP0061 100
T4 Polynucleotide Kinase, #EK0031 100
* 0.5 mM ATP (#R0441) is required for T4 DNA Ligase activity.

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