Qubit™ dsDNA BR Assay Kits - Ecologie Microbienne Lyon
Contents
1. 2 0 Fluorometer press DNA and then select dsDNA Broad Range as the assay type The Standards Screen is automatically displayed Note If you have already performed a calibration for the selected assay Qubit 2 0 Fluorometer will prompt you to choose between reading new standards and using the previous calibration See Calibrating the Qubit 2 0 Fluorometer above for calibration guidelines 1 10 On the Standards Screen press Yes to run a new calibration or press No to use the last calibration 1 11 Ifyou pressed No on the Standards Screen proceed to step 1 12 If you selected Yes to a run new calibration follow instructions below Running a New Calibration Insert the tube containing Standard 1 in the Qubit 2 0 Fluorometer close the lid and press Read The reading will take approximately 3 seconds Remove Standard 1 Insert the tube containing Standard 2 in the Qubit 2 0 Fluorometer close the lid and press Read Remove Standard 2 1 12 If you pressed No on the Standards Screen the Sample Screen will be automatically displayed Insert a sample tube into the Qubit 2 0 Fluorometer close the lid and press Read 1 13 Upon the completion of the measurement the result will be displayed on the screen Note The value given by the Qubit 2 0 Fluorometer at this stage corresponds to the concentration after your sample was diluted into the assay tube You can record this value and perform the calculation yourself to find2. e useful for quantitation of samples for microarray RT PCR and Northern blot procedures Qubit RNA BR Assay Kit Q10211 500 e accurate in the presence of DNA salts solvents proteins and free nucleotides Qubit RNA BR Assay Kit Q10210 100 RNA core range high confidence 1 25 g mL to 25 ug mLt Q33211 100 extended ranges moderate confidence 1 ug mL to 1 25 ug mL and 25 g mL to 26 ug mLt little protein to protein difference in signal e accurate in the presence of DTT B mercaptoethanol amino acids and DNA signal is stable for 3 hours Qubit Protein Assay Kit protein Qubit Protein Assay Kit Q33212 500 Based on an assay volume of 200 uL tConcentration ranges refer to the concentration of sample after dilution in the assay tube Qubit dsDNA BR Assay Kits 8 Product List current prices may be obtained from our website or from our Customer Service Department Cat no Product Name Unit Size Q32850 Qubit dsDNA BR Assay Kit 100 assays 2 1000 ng for use with the Qubit 2 0 Fluorometer tet 1 kit Q32853 Qubit dsDNA BR Assay Kit 500 assays 2 1000 ng for use with the Qubit 2 0 Fluorometer 0 0 c cece eee cence nee ee eens 1 kit Related products Q32851 Qubit dsDNA HS Assay Kit 100 assays 0 2 100 ng for use with the Qubit 2 0 Fluorometer 00 cece cece cece een e eee eeee 1 kit Q32854 Qubit dsDNA HS Assay Kit 500 assays 0 2
3. invitrogen MOLECULAR PROBES Qubit dsDNA BR Assay Kits For use with the Qubit 2 0 Fluorometer Table 1 Contents and storage information Material Amount Concentration Storage Stability Qubit dsDNA BR reagent 200X concentrate in Room temperature Component A 250 uL or 1 25 mL Desiccate Protect from light DMSO Qubit dsDNA BR buffer Component B 50 mL or 250 mL NA Room temperature When stored as directed kits are stable for 6 months Qubit dsDNA BR standard 1 Component C 1mLor5mL 0 ng uL in TE buffer Qubit dsDNA BR standard 2 Component D 1mLor5mL 100 ng uL in TE buffer NA Not applicable Introduction The Qubit dsDNA BR Assay Kits for use with the Qubit 2 0 Fluorometer make DNA quantitation easy and accurate The kit provides concentrated assay reagent dilution buffer and pre diluted DNA standards Simply dilute the reagent using the buffer provided add your sample any volume between 1 uL and 20 uL is acceptable and read the concentration using the Qubit 2 0 Fluorometer The assay is highly selective for double stranded DNA dsDNA over RNA Appendix Figure 1 and is accurate for initial sample concentration from 100 pg pL 1 000 ng uL The assay is performed at room temperature and the signal is stable for 3 hours Common contaminants such as salts free nucleotides solvents detergents or protein are well tolerated in the assay Appendix
4. Note When you navigate away from the Dilution Calculator screen the Qubit 2 0 Fluorometer saves the last values for the sample volume and the units in the Dilution Calculator screen only Returning to the Dilution Calculator screen displays these last selected values Qubit dsDNA BR Assay Kits 5 Appendix Selectivity of the Qubit dsDNA BR Assay DNA RNA 1 1 DNA Fluorescence RNA Aaa 0 200 400 600 800 1000 DNA or RNA ng Figure 1 DNA selectivity and sensitivity of the Qubit dsDNA BR assay Q32850 Q32853 Triplicate 10 uL samples of A DNA O E coli rRNA A or a 1 1 mixture of DNA and RNA W were assayed in the Qubit dsDNA BR assay Fluorescence was measured at 485 530 nm and plotted versus the mass of nucleic acid for the DNA alone or RNA alone or versus the mass of the DNA component in the 1 1 mixture The variation CV of replicate DNA determinations was lt 3 The inset a separate experiment with octuplicate determinations shows the sensitivity of the assay for DNA Background fluorescence has not been subtracted Effect of Temperature on the Qubit dsDNA BR Assay zT 160 5 T 140 2 Oo 2 Q 120 4 8 a 100 4 GE 29 80 z _ ge 60 a8 SN 40 co E 20 O 0 10 15 20 25 30 35 Assay temperature C Figure 2 Plot of fluorescence vs temperature for the Qubit dsDNA BR assay The Qubit assays were designed to be performed at room temperature
5. as temperature fluctuations can influence the accuracy of the assay Qubit dsDNA BR Assay Kits 6 How the Qubit 2 0 Fluorometer Calculates Concentration 120 Standards Raw Data Curve Fitting Algorithm Q f i 1 Relative fluorescence A O O oO to oO fi 0 1 2 3 4 5 6 Concentration of dsDNA g mL Figure 3 The curve fitting algorithm used to determine concentration in the Qubit dsDNA BR assay The Qubit 2 0 Fluorometer generates concentration data based on the relationship between the two standards used in calibration This plot shows the line corresponding to the curve fitting algorithm a modified Hill plot used in the calculation of concentration data for the Qubit dsDNA BR assay For reference the positions of the standards and a set of data points from an actual experiment are shown superimposed onto the line demonstrating that the curve fitting algorithm gives accurate values for quantitation Contaminants Tolerated by the Qubit dsDNA BR Assay Table 2 Effect of contaminants in the Qubit dsDNA BR assay tested over the range 0 01 g mL to 5 ug mL Contaminant Final Concentration Concentration in Concentration in Result in the Assay 20 uL Sample 10 pL Sample Sodium chloride 10 mM 100 mM 200 mM OKt Magnesium chloride 2mM 20 mM 40 mM OKt Sodium acetate 10 mM 100 mM 200 m
6. hour For this reason if you want to perform multiple readings of a single tube you should remove the tube from the instrument and let it equilibrate to room temperature for 30 seconds before taking another reading Calibrating the Qubit 2 0 Fluorometer For each assay you have the choice to run a new calibration or to use the values from the previous calibration As you first use the instrument you should perform a new calibration each time As you become familiar with the assays the instrument your pipetting accuracy and significant temperature fluctuations within your laboratory you should determine the level of comfort you have using the calibration data stored in from the last time the instrument was calibrated Remember also that the fluorescence signal in the tubes containing standards and the samples is stable for no longer than 3 hours See Figure 3 in the Appendix for an example of the calibration curve used to generate the quantitation results Qubit dsDNA BR Assay Kits 2 Handling and Disposal Experimental Protocol Performing the Qubit dsDNA BR Assay 1 1 1 2 1 3 1 4 1 5 1 6 1 7 We must caution that no data are available addressing the mutagenicity or toxicity of the Qubit dsDNA BR reagent Component A This reagent is known to bind nucleic acid and is provided as a solution in DMSO Treat the Qubit dsDNA BR reagent with the same safety precautions as all other potential mutagens and d
7. out the concentration of your original sample see Calculating the Concentration of Your Sample below or the Qubit 2 0 Fluorometer performs this calculation for you see Dilution Calculator next page 1 14 To read the next sample remove the sample from the Qubit 2 0 Fluorometer insert the next sample and press Read Next Sample 1 15 Repeat sample readings until all samples have been read Calculating the Concentration of Your Sample The Qubit 2 0 Fluorometer gives values for the Qubit dsDNA BR assay in g mL This value corresponds to the concentration after your sample was diluted into the assay tube To calculate the concentration of your sample use the following equation Concentration of your sample QF value x 0 x where QF value the value given by the Qubit 2 0 Fluorometer x the number of microliters of sample you added to the assay tube This equation generates a result with the same units as the value given by the Qubit 2 0 Fluorometer i e if the Qubit 2 0 Fluorometer gave a concentration in pg mL the result of the equation will be in ug mL Qubit dsDNA BR Assay Kits 4 Dilution Calculator 2 1 2 2 2 3 2 4 2 5 2 6 The Dilution Calculator feature of the Qubit 2 0 Fluorometer calculates the concentration of your original sample based on the volume of sample you have added to the assay tube To have the Qubit 2 0 Fluorometer perform this calculation for you follow
8. samples accurate in the presence of RNA salts solvents proteins and free Qubit dsDNA BR Assay Kit Q32853 500 nucleotides Qubit dsDNA BR Assay Kit Q32850 100 dsDNA core range high confidence 1 ng mL to 500 ng mLt extended ranges moderate confidence 0 5 ng mL to 1 ng mL and 500 ng mL to 600 ng mLt useful for quantitation of PCR products viral DNA and samples for subcloning Qubit dsDNA HS Assay Kit Q32854 500 accurate in the presence of RNA salts solvents proteins and free nucleotides Qubit dsDNA HS Assay Kit Q32851 100 dsDNA core range high confidence 5 ng mL to 1 000 ng mLt extended ranges moderate confidence 1 ng mL to 5 ng mL and 1 000 ng mL to 1 200 ng mLt useful for quantitation of oligos primers denatured DNA PCR products accurate in the presence of salts urea solvents proteins ATP and agarose Qubit ssDNA Assay Kit Q10212 100 ssDNA core range high confidence 25 ng mL to 500 ng mLt extended ranges moderate confidence 20 ng mL to 25 ng mL and 500 ng mL to 1 000 ng mLt e useful for quantitation of samples for microarray RT PCR and Northern blot procedures e accurate in the presence of DNA salts solvents proteins and free Qubit RNA Assay Kit Q32855 500 nucleotides Qubit RNA Assay Kit Q32852 100 RNA core range high confidence 0 1 ug mL to 5 ug mLt extended ranges moderate confidence 0 05 g mL to 0 1 g mL and 5 6 ug mLt
9. 100 ng for use with the Qubit 2 0 Fluorometer 0 cece eee e cence eee eee ee 1 kit Q10212 Qubit ssDNA Assay Kit 100 assays 1 200 ng for use with the Qubit 2 0 Fluorometer 2 ce cece cece cence tent eee eeee 1 kit Q10210 Qubit RNA BR Assay Kit 100 assays 20 1000 ng for use with the Qubit 2 0 Fluorometer 0 c cece eee cence eee eens 1 kit Q10211 Qubit RNA BR Assay Kit 500 assays 20 1000 ng for use with the Qubit 2 0 Fluorometer 00 cee eee eee cence nee ee eens 1 kit Q32852 Qubit RNA Assay Kit 100 assays 5 100 ng for use with the Qubit 2 0 Fluorometer 22 0 2 0 cece eee eee eect e een eeeneeee 1 kit Q32855 Qubit RNA Assay Kit 500 assays 5 100 ng for use with the Qubit 2 0 Fluorometer 0 cece eee c eee e eee cent ene e en eeee 1 kit Q33211 Qubit Protein Assay Kit 100 assays 0 25 5 ug for use with the Qubit 2 0 Fluorometer 0 cece cece eee cent nee ee eens 1 kit Q33212 Qubit Protein Assay Kit 500 assays 0 25 5 ug for use with the Qubit 2 0 Fluorometer 0 cee eee eee e cee eee ene eee eeee 1 kit Q32856 Qubit assay tubes setof 500 perisian aa rea REEE EE AEAEE ees hovese EERTE EA NEEE EEEE A 1 set Q32866 Qubit 2 0 FIUOroMetE sss sss iersirris sororis tris r ESPES EESE EDEA DOTEE SINE PITATE EEEIEE ERE EREKE ESEE EEEE EEEE SEERE TESA each Q32867 OQubit 20 Fluorometer USB isrisisererenere ren eea a OEE AA
10. E a E NAE A a sa EE EE each Q32868 Qubit 2 0 Fluorometer International Power Cord replacement 2 6 cece cece nen titer nee n een e eee e cnet en een eens each Contact Information Molecular Probes Inc 29851 Willow Creek Road Eugene OR 97402 Phone 541 465 8300 Fax 541 335 0504 Customer Service 6 00 am to 4 30 pm Pacific Time Phone 541 335 0338 Fax 541 335 0305 probesorder invitrogen com Toll Free Ordering for USA Order Phone 800 438 2209 Order Fax 800 438 0228 Technical Service 8 00 am to 4 00 pm Pacific Time Phone 541 335 0353 Toll Free 800 438 2209 Fax 541 335 0238 probestech invitrogen com Invitrogen European Headquarters Invitrogen Ltd 3 Fountain Drive Inchinnan Business Park Paisley PA4 ORF UK Phone 44 0 141 814 6100 Fax 44 0 141 814 6260 Email euroinfo invitrogen com Technical Services eurotech invitrogen com Further information on Molecular Probes products including product bibliographies is available from your local distributor or directly from Molecular Probes Customers in Europe Africa and the Middle East should contact our office in Paisley United Kingdom All others should contact our Technical Service Department in Eugene Oregon Molecular Probes products are high quality reagents and materials intended for research purposes only These products must be used by or directly under the supervision of a technically qualified individual ex
11. M OK Ammonium acetate 10 mM 100 mM 200 mM OKt ras phosphate 5 mM 50 mM 100 mM OKt Ethanol 1 10 20 OK Phenol 0 1 1 2 OK Chloroform 0 2 2 4 OK SDS 0 01 0 1 0 2 OK Triton X 100 0 001 0 01 0 02 OKt dNTPs 100 uM 1mM 2mM OK BSA 20 ug mL 200 ug mL 400 ug mL OKt IgG 10 ug mL 100 g mL 200 pg mL OK RNA 6X 6X 6X OK ssDNA 1X 1X 1X OK Oligos 3X 3X 3X OK DNA standards were assayed in the presence or absence of contaminants at the indicated final concentrations Equivalent concentrations approximate in 20 uL or 10 uL sample volumes are also listed In all cases results are given as OK usually less than 10 perturbation t An acceptable result but with some distortion of the standard curve for best results add the same amount of contaminant to the standard samples Immiscible A mixture of dATP dCTP dGTP and dTTP Qubit dsDNA BR Assay Kits 7 Qubit Assay Kits Compatible with the Qubit 2 0 Fluorometer A number of fluorescence based quantitation kits are available for use with the Qubit 2 0 Fluorometer Table 3 will help you choose a kit based on the target molecule being measured and the number of assays you require Table 3 Qubit Assay Kits for use with the Qubit 2 0 Fluorometer Product Cat no Numbero i Target Notes Assays core range high confidence 0 01 ug mL to 5 ug mLt extended range moderate confidence 5 ug mL to 10 ug mLt useful for quantitation of genomic and miniprep DNA
12. Table 2 In addition to the Qubit dsDNA BR Assay Kits described here we offer other kits for assaying RNA protein and dsDNA at a lower concentration range Appendix Table 3 To determine the purity of your sample use the Qubit dsDNA BR Assay Kit together with the Qubit RNA Assay Kit Thease measurements will give you a much better indication of sample purity than that produced by an A4 9 Ag measurement To measure protein contamination in nucleic acid samples simply run 1 20 uL of the sample in the Qubit protein assay Note All Qubit assay kits can also be used with the Qubit 1 0 Fluorometer MANO0002325 Revised 1 October 2010 MP 32850 Before You Begin Materials Required but Not Provided Storing the Qubit dsDNA BR Assay Kits Critical Assay Parameters e Plastic container disposable for mixing the Qubit working solution e Qubit assay tubes 500 tubes Cat no Q32856 or Axygen PCR 05 C tubes VWR part no 10011 830 The Qubit dsDNA BR reagent and buffer are designed for room temperature storage The Qubit dsDNA BR reagent is supplied in DMSO which freezes at temperatures lower than room temperature Store the DNA standards at 4 C Assay Temperature The Qubit dsDNA BR assay for the Qubit 2 0 Fluorometer delivers optimal performance when all solutions are at room temperature 22 28 C The Qubit assays were designed to be performed at room temperature as temperatur
13. e fluctuations can influence the accuracy of the assay Appendix Figure 2 To minimize temperature fluctuations store the Qubit dsDNA BR reagent and the Qubit dsDNA BR buffer at room temperature and insert all assay tubes into the Qubit 2 0 Fluorometer only for as much time as it takes for the instrument to measure the fluorescence because the Qubit 2 0 Fluorometer can raise the temperature of the assay solution significantly even over a period of a few minutes Do not hold the assay tubes in your hand before reading as this will warm the solution and result in a low reading Incubation Time To allow the Qubit assay to reach maximum fluorescence incubate the tubes for the DNA and RNA assays for 2 minutes after mixing the sample or standard with the working solution After this incubation period the fluorescence signal is stable for 3 hours at room temperature Photobleaching of the Qubit Reagent The Qubit reagents exhibit high photostability in the Qubit 2 0 Fluorometer showing lt 0 3 drop in fluorescence after 9 readings and lt 2 5 drop in fluorescence after 40 readings It is important to remember however that if the assay tube remains in the Qubit 2 0 Fluorometer for multiple readings a temporary reduction in fluorescence will be observed as the solution increases in temperature see Appendix Figure 2 The temperature inside the Qubit 2 0 Fluorometer may be as much as 3 C above room temperature after 1
14. ice For research use only Not intended for any animal or human therapeutic or diagnostic use Qubit dsDNA BR Assay Kits 9
15. ion or data 3 use of the product or its components for therapeutic diagnostic or prophylactic purposes or 4 resale of the product or its components whether or not such product or its components are resold for use in research Invitrogen Corporation will not assert a claim against the buyer of infringement of the above patents based upon the manufacture use or sale of a therapeutic clinical diagnostic vaccine or prophylactic product developed in research by the buyer in which this product or its components was employed provided that neither this product nor any of its components was used in the manufacture of such product If the purchaser is not willing to accept the limitations of this limited use statement Invitrogen is willing to accept return of the product with a full refund For information on purchasing a license to this product for purposes other than research contact Molecular Probes Inc Business Development 29851 Willow Creek Road Eugene OR 97402 Tel 541 465 8300 Fax 541 335 0504 Several Molecular Probes products and product applications are covered by U S and foreign patents and patents pending All names containing the designation are registered with the U S Patent and Trademark Office The trademarks mentioned herein are the property of Life Technologies Corporation or their respective owners Copyright 2010 Life Technologies Corporation All rights reserved This information is subject to change without not
16. ispose of the dye in accordance with local regulations The protocol below assumes you will be preparing standards for calibrating the Qubit 2 0 Fluorometer If you plan to use the last calibration performed on the instrument you will need fewer tubes step 1 1 and less working solution step 1 3 More detailed instructions on the use of the Qubit 2 0 Fluorometer corresponding to steps 1 9 1 15 and 2 1 2 6 can be found in the user manual accompanying the instrument For sample purity determinations it is possible to use the Qubit 2 0 Fluorometer to calculate the amount of dsDNA and RNA in the same sample Simply perform each assay for your sample Set up the number of 0 5 mL tubes you will need for standards and samples The Qubit dsDNA BR assay requires 2 standards Note Use only thin wall clear 0 5 mL PCR tubes Acceptable tubes include Qubit assay tubes 500 tubes Cat no Q32856 or Axygen PCR 05 C tubes VWR part no 10011 830 Label the tube lids Make the Qubit working solution by diluting the Qubit dsDNA BR reagent 1 200 in Qubit dsDNA BR buffer Use a clean plastic tube each time you make Qubit working solution Do not mix the working solution in a glass container Note The final volume in each assay tube must be 200 uL Each standard tube will require 190 uL of Qubit working solution and each sample tube will require anywhere from 180 uL to 199 uL Prepare sufficient Qubit working solutio
17. n to accommodate all standards and samples For example for 8 samples prepare enough working solution for the samples and 2 standards 200 uL per tube in 10 tubes yields 2 mL of working solution 10 uL of Qubit reagent plus 1 990 uL of Qubit buffer Load 190 uL of Qubit working solution into each of the tubes used for standards Add 10 uL of each Qubit standard to the appropriate tube and mix by vortexing 2 3 seconds being careful not to create bubbles Note Careful pipetting is critical to ensure that exactly 10 uL of each Qubit dsDNA BR standard is added to 190 uL of Qubit working solution It is also important to label the lid of each standard tube correctly as calibration of the Qubit 2 0 Fluorometer requires that the standards be introduced to the instrument in the right order Load Qubit working solution into individual assay tubes so that the final volume in each tube after adding sample is 200 uL Note Your sample can be anywhere between 1 uL and 20 uL therefore load each assay tube with a volume of Qubit working solution anywhere between 180 uL and 199 uL Add each of your samples to assay tubes containing the correct volume of Qubit working solution prepared in step 1 6 and mix by vortexing 2 3 seconds The final volume in each tube should be 200 uL Qubit dsDNA BR Assay Kits 3 1 8 Allow all tubes to incubate at room temperature for 2 minutes 1 9 On the Home Screen of the Qubit
18. perienced in handling potentially hazardous chemicals Please read the Material Safety Data Sheet provided for each product other regulatory considerations may apply Limited Use Label License No 223 Labeling and Detection Technology For research use only Not intended for any animal or human therapeutic or diagnostic use The purchase of this product conveys to the buyer the non transferable right to use the purchased amount of the product and components of the product in research conducted by the buyer whether the buyer is an academic or for profit entity The buyer cannot sell or otherwise transfer a this product b its components or c materials made using this product or its components to a third party or otherwise use this product or its components or materials made using this product or its components for Commercial Purposes The buyer may transfer information or materials made through the use of this product to a scientific collaborator provided that such transfer is not for any Commercial Purpose and that such collaborator agrees in writing a to not transfer such materials to any third party and b to use such transferred materials and or information solely for research and not for Commercial Purposes Commercial Purposes means any activity by a party for consideration and may include but is not limited to 1 use of the product or its components in manufacturing 2 use of the product or its components to provide a service informat
19. the instruction below Upon completion of the sample measurement press Calculate Stock Conc The Dilution Calculator Screen containing the volume roller wheel is displayed Using the volume roller wheel select the volume of your original sample that you have added to the assay tube When you stop scrolling the Qubit 2 0 Fluorometer calculates the original sample concentration based on the measured assay concentration To change the units in which the original sample concentration is displayed press ng mL A pop up window showing the current unit selection as indicated by an adjacent red dash opens Select the unit for your original sample concentration by touching the desired unit in the unit selection pop up window To close the unit selection pop up window touch anywhere on the screen outside the pop up The Qubit 2 0 Fluorometer automatically converts the units to your selection once the unit selection pop up window is closed Note The unit button next to your sample concentration reflects the change in the units e g if you change the unit to pg L the button will display pg L To save the data from your calculation to the Qubit 2 0 Fluorometer press Save on the Dilution Calculator screen The last calculated value of your measurement will be saved as a CSV file and tagged with a time and date stamp To exit the Dilution Calculator screen press any navigator button on the bottom of the screen or Read Next Sample
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