DNaseAlert User Manual
Contents
1. Read your assay immediately to limit exposure to UV light Substrate degradation The DNaseAlert Substrate contains DNA bases that will be depurinated and degraded when exposed to acidic conditions Substrate stimulation Certain organic solvents disrupt quenching In particular DNaseAlert Substrate always glows in acetonitrile Avoid use of solutions with pH lt 4 5 DNaseAlert Substrate Nuclease Detection System 62. If the tube remains clear the assay is negative and the sample is free of detectable DNase contamination If the tube glows pink DNase contamination is present A longwave 365 run UV source can be used but sensitivity will be lower Important safety note Never look directly into a UV light source Always use protective face shielding Quantitative measurement Place contents in a nuclease free cuvette or microtiter plate Measure fluorescence using a fluorometer set to the HEX channel 536 nm excitation 556 nm emission After the incubation at Step 4 above is complete the sample can be diluted with up to 2 mL Nuclease Free Water as needed to accommodate the size of the detection chamber Validate all negative assays Add 1 uL of DNase or other DNase to each tube Mix and incubate at 37 C for 10 min Repeat Step 5 Perform detection All negative tubes should now be positive Any assays that fail to fluoresce during this validation step must be considered failed and the nuclease detection assay should be repeated See troubleshooting guide at the end of this manual for help with assay failures Single use tubes testing a solid object or dry surface Set up one DNaseAlert assay for each sample to be tested Include 2 additional tubes for the positive and negative controls 1 Add 5 uL of Nuclease Free Water provided to a DNaseAlert Substrate single use tube for each assay Add 5 ul of 10X DNaseAlert B
3. Use gloves when handling kit components and performing the assay e Perform all steps under nuclease free conditions Use nuclease free pipette tips and tubes If necessary clean pipettes and other lab surfaces with Nuclease Decontamination Solution before use Single use tubes testing a liquid solution Set up one DNaseAlert assay for each sample to be tested Include 2 additional tubes for the positive and negative controls 1 Add 5 L of Nuclease Free Water provided to a DNaseAlert Substrate single use tube for each assay 2 Add 5 uL of 10X DNaseAlert Buffer to each tube 3 Add 40 pL of test sample to each sample tube Add 40 uL of Nuclease Free Water to each control tube Add 1 uL of DNase to the positive control tube Mix thoroughly The final concentration of substrate is 1 uM for the visual assay lower substrate concentrations can be used for fluorometric detection see Quantitative measurement below 4 Incubate at 37 C for 30 60 min Greater sensitivity is achieved with longer incubations If a temperature regulated incubator or water bath is not available incubation can be done at room temperature 2 3X longer incubation time is recommended to achieve similar sensitivity See what we can do for you at www idtdna com protocol molecular biology reagents Perform detection Quick visual confirmation Place tube on a shortwave 300 nm UV transilluminator For best results use a darkened room
4. eat detection procedure as before All negative tubes should now be positive Any assays that fail to fluoresce during this validation step must be considered failed and the nuclease detection assay should be repeated See troubleshooting guide at the end of this manual for help with assay failures 5 See what we can do for you at www idtdna com protocol Troubleshooting molecular biology reagents Observation False Negatives False Positives Possible Problem Presence of DNase inhibitors Solutions with extreme pH strong ionic strength or detergents can block action of DNases preventing detection of contaminants that are present Solution Test your solution for inhibitors of the DNaseAlert assay Set up a standard assay with your test solution and add 1 uL DNase or other DNase If the assay does not convert to positive after a 1 hour incubation your test solution is incompatible with the DNaseAlert system Low pH solutions Solutions with pH lt 7 0 will decrease the efficiency of HEX fluorescence lowering assay sensitivity If possible adjust the pH of the test solution to obtain pH gt 7 0 and lt 9 0 Substrate loss 1 The DNaseAlert Substrate is provided dried down The dry pellet can become dislodged from the bottom of the tube and may be lost from the tube when opened dry oligos can be electrostatically attracted to laboratory gloves Prolonged exposure of the DNaseA
5. help with assay failures Bulk substrate testing a liquid solution Bulk substrate assays can be performed in individual tubes however it is more convenient to perform the assays in microtiter plates We recommend using black opaque plates that minimize scatter and cross talk between wells DNaseAlert Always include negative and positive control wells Use duplicate or triplicate sample wells for quantitative measurements Substrate Nuclease Detection System 1 Add 1 mL of Nuclease Free Water provided to the bulk DNaseAlert Substrate tube Mix thoroughly Final substrate concentration is 2 uM 20 pmol substrate in 10 uL 2 Add 10 uL of DNaseAlert Substrate to each sample and control well 3 Add 10 uL of 10X DNaseAlert Buffer to each well 4 Add 80 uL of sample to the sample wells Add 80 pL of Nuclease Free Water to each control well Add 1 uL of DNase to the positive control well Mix thoroughly 5 Incubate at 37 C for 30 60 min The recommended final concentration of substrate is 200 nM for a standard 96 well fluorometer more dilute solutions can be used for sensitive cuvette fluorometers 6 Read assay in fluorometer using the HEX channel 536 nm excitation 556 nm emission Assays can be read as simple endpoint measurements or examined in real time to obtain quantitative kinetic curves 7 Validate all negative assays Add 1 uL of DNase or other DNase to each well Mix and incubate at 37 C for 10 min Rep
6. lert Substrate to light can lead to photobleaching of the fluorescent dye and decrease assay sensitivity 1 a Spin down tubes before opening b Perform a positive control assay Always perform a positive control test on single use tube assays with negative results A positive result in another tube even from the same kit is not sufficient 2 Store bulk substrate and assay tubes in the dark Nuclease substrate incompatibility The DNaseAlert Substrate contains a fluorescent dye at the 5 end and a fluorescence quencher at the 3 end Activity of some exonucleases is blocked by terminal end groups and therefore such nucleases e g E coli Exo I cannot be detected using this assay Contamination Nuclease contamination of tubes pipette tips and other lab equipment can lead to false positives Quencher exhaustion Prolonged exposure of the substrate to UV light can damage the quencher A visual assay left on an intense shortwave 254 nm UV source can turn positive even when no nuclease is present Perform a negative control assay A negative control must be included with each set of assays performed Fluorescence in the negative control tube Nuclease Free Water test indicates contamination in the experimental setup Obtain new supplies and clean work area If contamination is suspected obtain fresh tubes and pipette tips and clean all lab surfaces with Nuclease Decontamination Solution before repeating the assay
7. molecular biology reagents protocol DNaseAlert Substrate Nuclease Detection System RN User Manual e e T rar cx IDT See what we can do for you at www idtdna com a 14 H o a Oo 4S protocol EA Introduction molecular biology reagents Nucleases are widely present in the laboratory environment and can interfere with many experiments IDT developed DNaseAlert and RNaseAlert Substrate Nuclease Detection Systems for rapid sensitive detection of DNases and RNases respectively These reagents are fluorescence quenched oligonucleotide probes that fluoresce after nuclease degradation The assays can be read visually or measured and quantified using a fluorometer Assays can be used qualitatively or quantitatively to test lab reagents work surfaces equipment and supplies for nuclease contamination The reagents can also be used as highly sensitive substrates for enzyme kinetic studies The DNaseAlert Substrate is a synthetic DNA oligonucleotide that has a HEX reporter dye hexachlorofluorescein R on one end and a dark quencher Q on the other end Figure 1 Its sequence has been carefully optimized to react with a wide variety of nucleases it contains domains that will react with single stranded endonucleases single stranded exonucleases and double stranded nucleases Intact the substrate has little or no fluorescence When cleaved by a DNase the substrate fluoresces pink 536 nm
8. or UV excitation 556 nm emission The DNaseAlert test is fast and simple Lab surfaces or liquid reagents can be tested and verified as being nuclease free or contaminated in less than one hour For speed and ease of use a simple tube based visual assay can be performed directly at the site of testing Alternatively quantitative fluorescent results cuvette or microtiter plate formats can be obtained and used to document nuclease testing for GLP or manufacturing needs i po naO Bright Positive Assay Probe Cleaved DNase Contamination Present R DNA Q Incubate with Test Sample N QR DNA Q Dark Negative Assay Probe Intact DNase Contamination Absent Figure 1 DNaseAlert Assay DNaseAlert Substrate Nuclease Detection System 2 molecular biology reagents protocol DNaseAlert Substrate Nuclease Detection System Kit Components and Storage Conditions Component Quantity Storage Condition 25 single use tubes 50 pmol 20 C protect from DNaseAlert Substrate per tube or 2 tubes bulk light to prevent substrate 2 nmol per tube photobleaching DNaseAlert Buffer 250 uL 20 C Nuclease Free Water 2 mL Room temperature DNase Enzyme positive control 25 uL 20 C Nuclease Decontamination Solution 50 mL Room temperature Reagent quality is guaranteed for 6 months from date received when stored as directed Before you start e
9. uffer to each tube Add 40 uL of Nuclease Free Water to each tube Mix thoroughly Place the object to be tested into a sample tube Add 1 pL of DNase to the positive control tube e Pipette tips electrodes or other small solids can be dipped directly into the assay solution e To test surfaces that cannot be dipped into an assay tube a Wipe the surface of interest with a piece of nuclease free filter paper pre wetted with Nuclease Free Water b Soak the filter paper in a small amount of Nuclease Free Water c Transfer the liquid to an assay tube and proceed as instructed in A Single use tubes testing liquid samples DNaseAlert Substrate Nuclease Detection System 4 molecular biology reagents protocol 5 Incubate at 37 C for 30 60 min 6 Read assay in a fluorometer using the HEX channel 536 nm excitation 556 nm emission After Step 5 incubation is complete the sample can be diluted using up to 2 mL Nuclease Free Water as needed to accommodate the size of the detection chamber 7 Validate all negative assays Add 1 uL of DNase or other DNase to each negative non fluorescent tube Mix and incubate at 37 C for 10 min Repeat detection procedure as before All negative tubes should now be positive Any assays that fail to fluoresce during this validation step must be considered failed and the nuclease detection assay should be repeated See troubleshooting guide at the end of this manual for
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