Thermo Scientific™ FastDigest Buffer (10X)

Catalog No.	Quantity
FERB64	5 x 1 mL

Catalog No. FERB64 Supplier Thermo Scientific™ Supplier No. B64

Description

FastDigest Buffer (10X)

The 10X FastDigest Buffer is a proprietary digestion buffer which supports 100% activity of all FastDigest restriction enzymes. DNA/RNA modifying enzymes, such as ligases, phosphatases, kinases and mesophilic DNA polymerases also have 100% activity in FastDigest Buffer. Therefore, enzymes used in downstream applications can be directly added to the FastDigest reaction mix

Benefits:

Double and multiple digestion in a single buffer with any combination of enzymes.

No sequential digestions or buffer changes needed.

Specifications

• Concentration: 10X
• Quantity: 5 x 1 mL
• Product Type: Digestion Buffer
• Research Category: Traditional Cloning
• Product Line: FastDigest

Frequently Asked Questions (FAQs)

Can I store the FastDigest Buffers at 4 degrees C?

The FastDigest Buffers can be stored at 4 degrees C for at least 6 months. For long-term storage until their expiration, we recommend storing the FastDigest and FastDigest Green Buffers at -20 degrees C.

How I can transform or calculate reaction numbers in the unit of FastDigest enzymes?

The concentration of FastDigest enzymes is proprietary information. The FastDigest enzyme activity is measured by FastDigest unit (FDU): 1 µL of enzyme (1 FDU) cleaves 1 µg of DNA substrate in 5-15 min at 37 degrees C in 20 µL of 1X FastDigest buffer.

Are the FastDigest and FastDigest Green Buffer available separately?

Yes, the FastDigest Buffer (Cat. No. B64) and the FastDigest Green Buffer (Cat. No. B71) can be purchased separately.

What is the difference between FastDigest Buffer and FastDigest Green Buffer?

The FastDigest Green Buffer 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.

What is the composition of the FastDigest and FastDigest Green Buffer?

The FastDigest and FastDigest Green Buffers are proprietary digestion buffers which support 100% activity of all FastDigest restriction enzymes.

What is the unit or activity of FastDigest Restriction Enzymes?

Activity of FastDigest enzymes is defined as 1 µL of FastDigest enzyme cleaves1 µg of substrate DNA in 5 to 15 minutes in 20 µL of FastDigest buffer. This is in contrast to activity of conventional restriction enzymes which is defined as 1 unit of enzyme hydrolyzes 1 µg of lambda DNA in 60 minutes in 50 µL of optimal buffer for an enzyme.

What is the concentration of Mg2+ in the FastDigest buffers?

The composition of the FastDigest buffers is proprietary.

Is BSA present in the buffers of Thermo Scientific restriction enzymes?

As an added convenience, BSA is included in the conventional restriction enzyme buffers, as well as the FastDigest universal buffer. This eliminates the need to add BSA in a separate step.

Do FastDigest restriction enzymes contain BSA?

Yes, FastDigest restriction enzymes contain BSA.

Can I double digest my DNA using a Thermo Scientific conventional restriction enzyme and a FastDigest restriction enzyme?

For optimal results with fast reaction and 100% buffer compatibility, we highly recommend using FastDigest restriction enzymes in double digestion. In certain cases however, it may be possible to perform double digestion using a mix of Thermo Scientific conventional and Fastdigest restriction enzymes. For specific recommendations, please contact our technical service with detailed information about the enzymes and DNA template you plan to use.

Why do you recommend only 2 µL of 10X Reaction Buffer when digesting unpurified PCR product in a 30 µL reaction?

We recommend only 2 µl 10X Buffer in digestion of unpurified PCR products in 30 ul since salts and ions from the PCR reaction would be carried over to the digestion reaction.

What are key factors promoting star activity?

Star activty may be contributed by:

• Prolonged incubation
• High enzyme concentration
• High glycerol concentration (usually 5% or higher)
• Small reaction volume

Unexpected DNA bands were observed on agarose gel electrophoresis after restriction digestion. What may have caused this?

Unexpected cleavage patterns may be caused by the following reasons:

• Star activity of the restriction enzyme: Make sure to follow the reaction recommendations as specified in the protocol. Star activity may be improved by changing several key factors such as decreasing the reaction time, increasing the reaction volume, and decreasing the enzyme amount.

• Partial or incomplete cleavage (incomplete restriction reaction): Efficiency of the enzyme can be improved by adding more enzyme, prolonging the reaction time, or purifying DNA samples to remove inhibitory contaminants.

• Contamination with non-specific endonucleases: Non-specific endonucleases may be introduced to the DNA sample and/or the enzyme from improper handling, pipetting, etc.

•Improper reaction setup: Mix the digestion reaction thoroughly.

What are possible reasons for incomplete/failed restriction digestion?

The main reason for DNA cleavage reaction failure is the presence of contaminating inhibitors in the template DNA (for example: phenol, chloroform, detergents, ethanol, excess salts, EDTA, etc.). The best way to troubleshoot is to perform control reactions:

1) negative control (experimental DNA in the reaction buffer without the restriction enzyme) to access degradation of DNA by contaminants in the DNA template and/or reaction buffer
2) positive control reaction I (digestion of highly pure control DNA with the restriction enzyme) to access reaction conditions and enzyme activity
3) positive control reaction II (highly pure control DNA + experimental DNA + Restriction Enzyme) to access possible issues with the experimental DNA.

In addition, please check for sensitivity of the restriction enzymes to template DNA methylation.

Is there any other way to inactivate Thermo Scientific restriction enzymes (when thermal inactivation is not applicable) without using phenol/chloroform?

To remove the enzymes without using phenol/chloroform, we would recommend silica column based purification such as GeneJET Gel Extraction and DNA Cleanup Micro Kit (Cat. Nos. K0831/2) or GeneJET PCR Purification Kit (Cat. Nos. K0701/2).

What is the stability of Thermo Scientific restriction enzymes if they freeze-thaw during shipment?

Enzymes may freeze during shipment on dry ice. This does not affect their quality as all Thermo Scientific enzymes are tested 100% active after at least three freeze-thaw cycles. For 24-48 hour delivery, enzymes may be shipped on blue ice because their quality is not affected by short exposure to 4 degrees C.

Do Thermo Scientific restriction enzymes come with the buffer(s)?

Thermo Scientific restriction enzymes are shipped with their optimal digestion buffer(s). The buffers are available separately for purchase as well.

For Research Use Only. Not for use in diagnostic procedures.