Qubit™ dsDNA HS Assay Kits - Ecologie Microbienne Lyon
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1. invitrogen MOLECULAR PROBES Qubit dsDNA HS Assay Kits For use with the Qubit 2 0 Fluorometer Table 1 Contents and storage information Material Amount Concentration Storage Stability Qubit dsDNA HS Reagent 5 Room temperature g 250 uL or 1 25 mL 200X concentrate in DMSO Desiccate Component A Protect from light Qubit dsDNA HS Buffer Component B 50 mL or 250 mL NA Room temperature When stored as directed kits are Qubit dsDNA HS Standard 1 Component C stable for 6 months 1mLor5 mL 0 ng uL in TE buffer e lt 4 C Qubit dsDNA HS Standard 2 Component D 1mLor5 mL 10 ng pL in TE buffer NA Not applicable Introduction MANO0002326 The Qubit dsDNA HS 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 concentrations from 10 pg uL to 100 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 protei2. 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 Set up the number of 0 5 mL tubes you will need for standards and samples The Qubit dsDNA HS 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 HS reagent 1 200 in Qubit dsDNA HS 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 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 solution 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
3. Qubit dsDNA HS 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 Allow all tubes to incubate at room temperature for 2 minutes Qubit dsDNA HS Assay Kit 3 1 9 On the Home Screen of the Qubit 2 0 Fluorometer press DNA and then select dsDNA High Sensitivity 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 Scre
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5. determine concentration in the Qubit dsDNA HS 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 HS 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 Table 2 Effect of contaminants in the Qubit dsDNA HS assay tested over the range of 1 ng mL to 500 ng mL Final TA RR Contaminant Concentration in concen earen at concen earlegls Result the Assay pL Sample 10 pL Sample Sodium chloride 50mM 500 mM 1M OK Magnesium chloride 5mM 50mM 100 mM OK t Sodium acetate 30 mM 300 mM 600 mM OK Ammonium acetate 50mM 500 mM 1M OK Ethanol 1 10 20 OK Phenol 0 1 1 2 OK t Chloroform 1 10 20 OK SDS 0 01 0 1 0 2 OK Triton X 100 0 01 0 1 0 2 OK t dNTPs 100 uM 1mM 2mM OK BSA 10 mg mL 100 mg mL 200 mg mL OKt IgG 0 5 mg mL 5 mg mL 10 mg mL OK RNA 1X 1X 1X OK ssDNA 1X 1X 1X OK Oligos 1X 1X 1X OK DNA standards were assayed in the presence or absence of contaminants at the indicated final concentrations Equivalent in 20 uL or 10 uL sample volumes are also listed In
6. the fluorescence as 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 In order to allow the Qubit assay to reach optimal 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 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 calibrat
7. 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 concentrations approximate Qubit dsDNA HS Assay Kit 7 Qubit Assay Kits Compatible with the Qubit 2 0 Fluorometer Table 3 Qubit Assay Kits for use 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 Product Cat no numberof Target Notes Assays Qubit dsDNA BR Assay Kit 032850 100 core range high confidence 0 01 ug mL to 5 ug mLt extended range moderate confidence 5 ug mL to 10 ug mLt dsDNA useful for quantitation of genomic and miniprep DNA samples e accurate in the presence of RNA salts solvents proteins and free Qubit dsDNA BR Assay Kit Q32853 500 nucleotides i core range high confidence 1 ng mL to 500 ng mLt Qubit dsDNA HS Assay Kit 032851 100 extended ranges moderate confidence 0 5 ng mL to 1 ng mL and 500 ng mL to 600 ng mLt dsDNA useful for quantitation of PCR products viral DNA and samples for subcloning Qubit dsDNA HS Assay Kit 032854 500 e accurate in the presen
8. ce of RNA salts solvents proteins and free nucleotides core range high confidence 5 ng mL to 1 000 ng mLt extended ranges moderate confidence 1 ng mL to 5 ng mL and en 1 000 ng mL to 1 200 ng mLt Qube ssDNA Assay Kit Q10212 109 SSDNA useful for quantitation of oligos primers denatured DNA PCR products e accurate in the presence of salts urea solvents proteins ATP and agarose core range high confidence 25 ng mL to 500 ng mLt Qubit RNA Assay Kit Q32852 100 extended ranges moderate confidence 20 ng mL to 25 ng mL and 500 ng mL to 1 000 ng mLt RNA useful for quantitation of samples for microarray RT PCR and Northern blot procedures accurate in the presence of DNA salts solvents proteins and free Qubit RNA Assay Kit Q32855 500 nucleotides Qubit RNA BR Assay Kit Q10210 100 Qubit RNA BR Assay Kit Q10211 500 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 useful for quantitation of samples for microarray RT PCR and Northern blot procedures accurate in the presence of DNA salts solvents proteins and free nucleotides Qubit Protein Assay Kit Q33211 100 Qubit Protein Assay Kit Q33212 500 protein core range high confidence 1 25 g mL to 25 ug mLt extended ranges moderate confidence 1 ug mL to 1 25 g mL and 25 g mL to 26 ug mLt little p
9. 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 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 HS Assay Kit 5 Appendix Selectivity of the Qubit dsDNA HS Assay DNA RNA 1 1 DNA 0 0 1 0 2 0 DNA ng Fluorescence A pA 0 20 40 60 80 100 DNA or RNA ng Figure 1 DNA selectivity and sensiti
10. e eee 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 e eee eect nee en eens 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 AAE 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 Serv
11. en 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 find 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 HS assay in ng mL This value corres
12. ice 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 experienced 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
13. ion 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 HS Assay Kit 2 Handling and Disposal Experimental Protocol 1 1 1 2 1 3 1 4 1 5 1 6 1 7 1 8 We must caution that no data are available addressing the mutagenicity or toxicity of the Qubit dsDNA HS reagent Component A This reagent is known to bind nucleic acid and is provided as a solution in DMSO Treat the Qubit dsDNA HS reagent with the same safety precautions as all other potential mutagens and dispose 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
14. n are well tolerated in the assay Appendix Table 2 In addition to the Qubit dsDNA HS Assay Kits described here we offer other kits for assaying RNA protein and dsDNA at a higher concentration range Appendix Table 3 Note All Qubit assay kits can also be used with the Qubit 1 0 Fluorometer Revised 1 October 2010 MP 32851 Before You Begin Materials Required but Not Provided Storing the Qubit dsDNA HS Assay Kits Critical Assay Parameters e Plastic container disposable for mixing the Qubit working solution step 1 3 e Qubit assay tubes 500 tubes Cat no Q32856 or Axygen PCR 05 C tubes VWR part no 10011 830 The Qubit dsDNA HS reagent and buffer are designed for room temperature storage The Qubit dsDNA HS 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 HS 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 temperature fluctuations can influence the accuracy of the assay Appendix Figure 2 To minimize temperature fluctuations store the Qubit dsDNA HS reagent and the Qubit dsDNA HS 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
15. ponds 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 2 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 cncentration in ng mL the result of the equation will be in ng mL Qubit dsDNA HS Assay Kit 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 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
16. rotein to protein difference in signal accurate in the presence of DTT 8 mercaptoethanol amino acids and DNA signal is stable for 3 hours Based on an assay volume of 200 uL tConcentration ranges refer to the concentration of sample after dilution in the assay tube Qubit dsDNA HS Assay Kit 8 Product List current prices may be obtained from our website or from our Customer Service Department Cat no Product Name Unit Size Q32851 Qubit dsDNA HS Assay Kit 100 assays 0 2 100 ng for use with the Qubit 2 0 Fluorometer Es 1 kit Q32854 Qubit dsDNA HS Assay Kit 500 assays 0 2 100 ng for use with the Qubit 2 0 Fluorometer 0 cece eee e cence nee ee eens 1 kit Related products Q32850 Qubit dsDNA BR Assay Kit 100 assays 2 1000 ng for use with the Qubit 2 0 Fluorometer 00sec cece cece ene eee eee 1 kit Q32853 Qubit dsDNA BR Assay Kit 500 assays 2 1000 ng for use with the Qubit 2 0 Fluorometer 0 cee cece cece ene eee eee 1 kit Q10212 Qubit ssDNA Assay Kit 100 assays 1 200 ng for use with the Qubit 2 0 Fluorometer ce cece eee e cence eee eee ee eens 1 kit Q10210 Qubit RNA BR Assay Kit 100 assays 20 1000 ng for use with the Qubit 2 0 Fluorometer 0 cece eee eee een 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 ec cece e ene en
17. rovided 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 notice For research use only Not intended for any animal or human therapeutic or diagnostic use Qubit dsDNA HS Assay Kit 9
18. vity of the Qubit dsDNA HS assay Q32851 Q32854 Triplicate 10 uL samples of A DNA O E coli rRNA A ora 1 1 mixture of DNA and RNA W were assayed in the Qubit dsDNA HS 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 2 The inset a separate experiment with octuplicate determinations shows the extreme sensitivity of the assay for DNA Background fluorescence has not been subtracted Effect of Temperature on the Qubit dsDNA HS Assay 160 140 120 4 100 80 4 60 4 40 Relative fluorescence normalized to room temperature 20 0 10 15 20 25 30 35 Assay temperature C Figure 2 Plot of fluorescence vs temperature for the Qubit dsDNA HS assay The Qubit assays were designed to be performed at room temperature as temperature fluctuations can influence the accuracy of the assay Qubit dsDNA HS Assay Kit 6 How the Qubit 2 0 Fluorometer Calculates Concentration Contaminants Tolerated by the Qubit dsDNA HS Assay Relative fluorescence 120 Standards 100 4 Raw Data Curve Fitting Algorithm 80 60 40 4 20 4 O 100 200 300 400 500 600 Concentration of dsDNA ng mL Figure 3 The curve fitting algorithm used to
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