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Cat. No.: DAFA-100 (for 100T)

Cat. No.: DAFA-500 (for 500T)

Description

DNase Activity Fluorometric Assay Kit, also known as the Fluorometric DNase Assay Kit, is a reagent kit used for rapid and highly sensitive detection of DNase I and other deoxyribonucleases (DNases) using a fluorescence-based method.

Similar to ribonucleases (RNases), DNases are widely present in experimental environments and biological organisms. The presence of nucleases can interfere with many experiments due to their ability to degrade nucleic acids. Common methods for detecting DNase in literature are often time-consuming and have relatively low detection sensitivity.

This assay kit offers high sensitivity and can detect DNase I activity as low as approximately 6×10-4 U. It is also referred to as the Deoxyribonuclease Activity Fluorometric Assay Kit, DNA Enzyme Activity Fluorometric Assay Kit, DNase I Enzyme Activity Fluorometric Assay Kit, Fluorometric DNase Activity Assay Kit, Fluorometric DNase I Activity Assay Kit, DNase Residual Assay Kit, DNA Enzyme Residual Assay Kit, or Deoxyribonuclease Residual Assay Kit.

The most common DNase is DNase I, also known as Deoxyribonuclease I. It is an endonuclease that can digest single-stranded or double-stranded DNA, producing single deoxyribonucleotides or single-stranded or double-stranded oligodeoxyribonucleotides. The hydrolysis products of DNase I have a phosphate group at the 5' end and a hydroxyl group at the 3' end. DNase I activity is calcium-dependent and can be activated by magnesium or divalent manganese ions. In the presence of magnesium ions, DNase I can randomly cleave double-stranded DNA at any site, while in the presence of divalent manganese ions, DNase I can cleave DNA double-strands at the same site, generating blunt ends or sticky ends with 1-2 nucleotides overhang.

The DNase Activity Fluorometric Assay Kit utilizes the principle of Fluorescence Resonance Energy Transfer (FRET). The DNase substrate is a synthetic DNA oligonucleotide probe, with a VIC fluorophore (donor) on one end and a BHQ1 quencher (acceptor) on the other end. These two fluorophores have partially overlapping absorption spectra. When the distance between the two fluorophores is suitable, fluorescence energy is transferred from the donor to the acceptor, resulting in a decrease in the fluorescence intensity of the donor fluorophore. VIC and BHQ1 are connected to both ends of the DNase substrate. When the substrate is cleaved by DNase, the ends of the DNA substrate separate, causing the two fluorophores to dissociate. The fluorescence of VIC is no longer quenched by BHQ1, allowing the detection of VIC fluorescence. Therefore, DNase enzyme activity can be detected with high sensitivity using fluorescence detection. The maximum excitation wavelength of VIC is 535nm, and the maximum emission wavelength is 556nm. The kit provides DNase I standard to establish a standard curve for calculating the enzyme activity of the samples.

Content

Cat. No.: DAFA-100 (for 100T)

• 10X Reaction Buffer        2ml
• DNase I (1U/μl)                 20μl
• 5X DNase Substrate     200μl
• Nuclease-free Water     20ml

Cat. No.: DAFA-500 (for 500T)

• 10X Reaction Buffer       10ml
• DNase I (1U/μl)                100μl
• 5X DNase Substrate         1ml
• Nuclease-free Water   100ml

Features

This assay kit has high detection sensitivity and requires a small sample volume. The kit includes DNase I as a positive control, which facilitates the establishment of the detection system. The DNase substrate probe has been optimized for high sensitivity, allowing detection of DNase I activity as low as approximately 6×10-4 U. The detection sensitivity of this kit is higher than that of conventional similar products. Users can calculate the activity of DNase I in their samples by setting up a standard curve.

This assay kit has a wide range of applications, is flexible to use, and provides fast detection. It can not only be used to detect DNase I but also other DNases, including nucleases that can cleave single-stranded and double-stranded DNA. Compared to ELISA methods, this kit offers a simpler, faster, and more accurate approach. Experimental validation has shown that this assay kit can be used to detect the relative activities of enzymes such as Benzonase-like super nucleases, T4 DNA polymerase, T5 Exonuclease, Micrococcal nuclease, Mung Bean Nuclease, and S1 Nuclease. The assay kit utilizes a one-step detection method, which is simple and rapid, with a total assay time of approximately 20 minutes.

When used for 96-well plate assays, the small packaging of this assay kit allows for 100 tests, while the medium packaging allows for 500 tests.

Precautions

• Due to the high sensitivity of this assay kit and the potential presence of deoxyribonucleases (DNases) in the environment, it is recommended to perform DNase activity detection in a relatively clean environment such as a laminar flow hood or biosafety cabinet to prevent interference from DNases present in the surroundings.
• The 10X Reaction Buffer, DNase Buffer, and 5X DNase Substrate should be completely thawed and equilibrated to room temperature before use; otherwise, it may affect the detection results. DNase I (1U/μl) should be kept on ice during use and all reagents should be immediately stored according to the kit's storage conditions after use.
• Ensure that the sample pH is between 7 and 8, or the pH of the reaction system after adding the sample is within this range; otherwise, it may affect the signal values and stability of the detection results. For small volume reagents, it is recommended to briefly centrifuge the vial to ensure the liquid settles at the bottom before use. Thawed reagents must be completely melted and mixed well before use.
• This assay kit may not be suitable for some nucleases such as Klenow Fragment and Phi29 DNA polymerase. In general, the provided 10X Reaction Buffer in this kit is compatible with most nucleases, but there may be specific cases where it is not suitable for certain enzymes. If necessary, dilute and react the sample with a specific nuclease buffer.
• To prevent contamination of the reagents with DNase, it is recommended to use RNase and DNase Away to remove DNase present in the environment before each experiment, if needed.
• For detection, it is recommended to use 96-Well Black Opaque Plates.
• This product is intended for scientific research by professionals only and should not be used for clinical diagnosis or treatment, food or drug purposes, or stored in regular residential areas.
• For your safety and health, please wear lab attire and disposable gloves during operation.

Storage

The minimum shelf life is 1 year at -20°C. The 5X DNase Substrate should be stored protected from light.

Related: RNase Activity Fluorometric Assay Kit