Product Specification Sheet
Contents
1. Compound Initial concentration Final concentration Decrease in DNA sample in assay 200 uL in Signal Sodium Chloride 1M 50 mM 14 Magnesium Chloride 100 mM 5 mM 16 Sodium Acetate 600 mM 30 mM 11 Ammonium Acetate 1M 50 mM 14 Ethanol 20 1 21 Phenol 2 0 10 11 Chloroform 20 1 34 SDS 0 2 0 01 31 SDS 0 02 0 001 9 Triton X 100 0 2 0 01 36 Triton X 100 0 02 0 001 20 Tween 20 0 1 0 005 20 BSA 20 mg mL 1 mg mL 36 dNTPs 2 mM 100 uM 11 12000 Calf Thymus 10000 4 a Lambda DNA a ssDNA RNA 8000 6000 Fluorescence 4000 2000 T T T 0 50 100 150 200 250 DNA ng well Figure 3 Selectivity of AccuClear dsDNA quantitation assay for double stranded DNA compared to single stranded DNA and single stranded RNA Related Products Catalog Product number E90001 AccuLite 470 Mini Fluorometer AccuClear Ultra High Sensitivity dsDNA Quantitation Solution 31027 1000 assays 31028 AccuClear Ultra High Sensitivity dsDNA Quantitation Kit with 7 DNA Standards 1000 assays 31006 AccuBlue High Sensitivity dsDNA Quantitation Kit 31007 AccuBlue Broad Range dsDNA Quantitation Kit 31008 T AccuBlue High Sensitivity dsDNA Quantitation Solution trial size 31009 T AccuBlue Broad Range dsDNA Quantitation Solution trial size 41003 GelRed Nucleic Acid Ge2. The two lowest concentration DNA dilutions H 1 should be prepared fresh on the day of assay The other DNA dilutions B G can be stored at 4 C for at least 6 months with the addition of 2 mM final concentration sodium azide Standard Concentration DNA EOI cEL buffer A 25 ng uL 31029C AccuClear DNA Standard 25 ng uL stock B 10 ng uL 400 uL of A 600 uL 0 3 ng uL 120 uL of A 880 uL D 1 ng uL 100 uL of B 900 uL E 0 3 ng uL 100 uL of C 900 uL F 0 1 ng uL 100 uL of D 900 uL G 0 03 ng uL 100 uL of E 900 uL H 0 01 ng uL 100 uL F 900 uL 0 003 ng uL 100 uL of G 900 uL J 0 ng uL OuL 1000 uL 5 On the day of the assay prepare working solution Dilute the dye at a ratio of 1 100 in buffer in a plastic container do not use a glass container and mix well by vortexing or shaking Prepare 200 uL of working solution for each sample to be tested For example mix 200 uL of dye with 20 mL 1X assay buffer to prepare enough working solution for an entire 96 well plate Volumes can be scaled as required Prepare only as much working solution as you plan to use within 24 hours and discard unused working solution 24 hours after mixing 6 For each sample to be tested pipette 200 uL of the working solution per well of a black 96 well microplate To test samples in triplicate prepare three separate wells for each DNA standard and three separate wells for each unknown DNA sample Accurate multi channel pipe
3. of AccuClear dye bound to dsDNA AccuClear Ultra High Sensitivity dsDNA Quantitation Kit with 1 DNA Standard PS006 Assay Protocol Note see the Appendix for information on using the AccuClear assay with the AccuLite 470 fluorometer 1 Use properly calibrated pipettes and DNase free pipette tips tubes and plates for best accuracy It is recommended to test each DNA standard and each unknown sample in triplicate If more than one 96 well plate is to be tested in a single assay it is recommended to include a standard curve on each plate to minimize variability between plates See Considerations for Data Analysis next page for more information on standards 2 Warm all components to room temperature before use AccuClear dye is provided in DMSO which may freeze during storage at 4 C You can place all kit components in a 37 C water bath for rapid warming be sure to allow solutions to cool to room temperature before using Before removing the required volume mix each component well by shaking or vortexing and centrifuge vials briefly before opening to minimize reagent loss on the cap 3 Prepare 1X AccuClear buffer by diluting the 20X buffer solution 1 20 in dH O 1X AccuClear buffer can be stored at 4 C for at least 6 months with the addition of 2 mM final concentration sodium azide 4 Prepare a set of DNA standards by diluting the 25 ng uL standard in 1X AccuClear buffer as shown below Volumes may be scaled as necessary
4. Biotlum Glowing Products for Science 3159 Corporate Place Hayward CA 94545 www biotium com Revised February 20 2014 Product Information AccuClear Ultra High Sensitivity dsDNA Quantitation Kit with 1 DNA Standard Catalog Number 31029 Unit Size 2000 assays 200 uL microplate assay Kit Contents Component Size 99977 AccuClear dye 100X in DMSO 4X1 mL 99979 20X AccuClear buffer 50 mL 31029C AccuClear DNA Standard 3X1 mL 25 ng uL calf thymus DNA in TE buffer Storage and Handling Store kit at 4 C Protect dye from light The kit is stable for at least 6 months from date of receipt when stored as recommended AccuClear dye is a potentially harmful chemical Exercise universal laboratory safety precautions when handling the dye and dispose of the dye as hazardous chemical waste according to your local regulations Spectral Properties Ex Em 468 507 nm bound to dsDNA See Figure 1 for spectra Product Description AccuClear Ultra High Sensitivity dsDNA Quantitation Kits provide highly sensitive and accurate DNA quantitation across a broad range of DNA concentrations Figure 2 The assay is linear between 30 pg and 250 ng of dsDNA per assay 3 pg uL to 25 ng uL sample concentration in microplate format Unlike absorbance based measurements AccuClear dsDNA Quantitation dye is highly selective for double stranded DNA over single stranded DNA or RNA Figure 3 The AccuClear Ultra Hig
5. e equation for the standard curve trend line to calculate the amount of unknown DNA in each well y fluorescence and x ng DNA per well Note the standard curve shown in Figure 2 is for reference only You must generate your own standard curve using your instrument to calculate the amount of DNA in your unknown samples Considerations for Data Analysis Calf thymus DNA can serve as a reference for most plant and animal DNA because it is double stranded highly polymerized and is approximately 58 AT 42 GC Lambda dsDNA yields similar results Figure 3 You may wish to use a standard similar to your unknown samples in DNA length structure i e linear vs circular or GC content For bacterial DNA a species specific standard may be desired because the GC content varies widely depending on the species The AccuClear dsDNA quantitation assay is available without standards catalog no 31027 for customer who wish to prepare their own standards The linear range of the AccuClear assay extends from 250 ng to 0 03 ng The standard curve can be extended to 300 ng with some loss of linearity If lower end standards are desired you can prepare 0 01 ng uL and 0 003 ng uL standards by diluting the 0 1 ng uL and 0 03 ng uL DNA 1 10 in AccuClear buffer Use 10 uL of these standards in the assay to obtain 0 1 ng and 0 03 ng data points It is recommended to prepare the 0 01 ng uL and 0 003 ng uL standards fresh on the day of assay If the fluoresc
6. ence of any of the unknown samples is higher than the linear range further dilute the sample and add 10 uL of the diluted sample to perform the assay For consistency it is best to use the same volume of sample in all the wells Due to differences in instruments check instrument settings to optimize for the best linearity Some factors that can affect the final linearity and relative fluorescence intensity are 1 the excitation and emission wavelengths and bandwidths 2 cut off filters 3 sensitivity settings 4 pipetting accuracy and 5 microplate manufacturer The effects of common DNA contaminants such as salts solvents detergents and protein on the AccuClear assay are listed in Table 1 Please also see our AccuBlue dsDNA Quantitation Assays related products which are more tolerant of some contaminants compared to AccuClear 14000 2 t 12000 to y a gt 10000 Fluorescence o 0 0 2 0 4 0 6 0 8 1 DNAng well 2 3S 3S 6000 Fluorescence 0 50 100 150 200 250 DNAng well Figure 2 Linearity of AccuClear dsDNA quantitation assay between 30 pg and 250 ng per well in microplate assay with excitation emission at 468 507 nm The inset shows the lower portion of the curve AccuClear Ultra High Sensitivity dsDNA Quantitation Kit with 1 DNA Standard PS006 Table 1 Effect of common DNA contaminants on AccuClear assay signal
7. er into the 0 ng DNA tube blank Pipette 10 uL of the 10 ng uL DNA standard into the 100 ng DNA tube Pipette up and down or vortex to mix 4 Prepare samples by pipetting 10 uL of each sample DNA per tube Pipette up and down or vortex to mix Calibration To move to a previous screen at any time select Return Continue selecting Return to go back to the Main Menu 1 From the AccuLite Main Menu select Calibrate 2 Select Accu dsDNA from the assay list 3 Insert the blank tube and close the cover Select Blank 4 After blanking the standard value will appear 00100 000 Insert the 100 ng DNA standard tube and close the cover Press Measure 5 Calibration Finished will appear on the screen 6 Select Return to return back to the Main Menu Sample Measurement 1 From the AccuLite Main Menu select Measure 2 SelectAccu dsDNA from the assay list 3 Insert the first sample tube and close the cover Select Measure The value shown is ng DNA per tube 4 Select Save to save the data in the meter Alternatively you can manually the record data without saving then select Return 5 Insert next sample and select Measure 6 After reading all samples select Return repeatedly to navigate back to main menu Retrieving Saved Data 1 From the AccuLite Main Menu select Data 2 Select Accu dsDNA from the assay list 3 Use the arrow keys to navigate through saved data points Data points are numbered in order o
8. f measurement 4 To erase data select Erase All and Confirm 5 To return to previous screens select Return Performing a Full Calibration Curve with AccuLite The first time you perform the assay or if unexpected results are obtained you may wish to perform a full calibration curve to verify that the assay is performing properly In this case perform the 2 point calibration as described above then read the full set of standards as if they were unknown samples Plot the standard curve as described in the AccuClear protocol AccuClear Ultra High Sensitivity dsDNA Quantitation Kit with 1 DNA Standard PS006 Main Menu Measure Calibrate Info Data x x 1 Select Calibrate 7 Select Measure 13 Select Data y t y Accu dsDNA Accu dsDNA Accu dsDNA AccuOrange AccuOrange AccuOrange Assay 3 Assay 3 Assay 3 Return Return Return 2 Select Accu dsDNA 8 Select Accu dsDNA 14 Select Accu dsDNA t t y Accu dsDNA Accu dsDNA Data Blank lt HH gt Blank Measure Erase All Return Return Return 3 Insert blank tube 9 Insert sample tube 15 Use arrows to scroll select Blank select Measure through data points Or J J Select Erase All 00100 000 Timer y Erase All lt Std gt Data Value lt lt Measure Save Return Return Confirm 4 Standard value is 10 Select Save or displayed 000100 000 Return if not savin
9. g Return 16 Select Return repeatedly to return to Timer Main Menu 5 Insert 100 ng standard Data Value tube select Measure Measure 1 Return 4 1 Insert next sample Calibration tube select Measure Finished Repeat for each sample Return 12 Select Return repeatedly to return to 6 Select Return once to Main Menu return to Main Menu Figure 4 AccuLite user menu tree showing AccuClear calibration measurement and data retrieval steps See the AccuLite user manual for complete user menu tree Page 3 of 3
10. h Sensitivity dsDNA quantitation assay is designed for use with fluorescence 96 well plate readers equipped with excitation and emission filters for detecting green fluorescence The unique spectral properties of AccuClear dye make it especially well suited for use with instruments with blue LED excitation sources Biotium s AccuLite 470 handheld fluorometer is pre programmed for use with the AccuClear assay AccuClear also is compatible with handheld fluorometers such as Invitrogen s Qubit and Promega s QuantiFluor P however the standard curve calibration programs for these instruments may not cover the full dynamic range of the AccuClear kit standard curve The AccuClear Ultra High Sensitivity dsDNA Quantiation Kit with 1 DNA Standard contains AccuClear dye 20X assay buffer and a 25 ng uL calf thymus dsDNA standard stock solution that can be used to prepare a set of standards Biotium also offers the AccuClear Ultra High Sensitivity dsDNA Quantiation Kit with 7 DNA Standards 1000 assays catalog no 31028 which includes AccuClear dye 1X assay buffer and a set of prediluted calf thymus dsDNA standards AccuClear Ultra High Sensitivity dsDNA Quantitation Solution 1000 assays catalog no 31027 which includes dye and 1X buffer without standards is available for customers who wish to prepare their own DNA standards Absorbance uolss w3 300 400 500 600 700 Wavelength nm Figure 1 Absorbance and emission spectra
11. l Stain 10 000X in water 31003 T Fast EvaGreen qPCR Master Mix trial size 31020 T Fast Plus EvaGreen qPCR Master Mix trial size Please visit our website at www biotium com for information on our life science research products including environmentally friendly EvaGreen qPCR master mixes fluorescent CF dye antibody conjugates and reactive dyes apoptosis reagents fluorescent probes and kits for cell biology research AccuBlue AccuClear and AccuLite are trademarks of Biotium Inc Qubit is a registered trademark of Molecular Probes Inc QuantiFluor is a trademark of Promega Corp AccuBlue technology is covered by U S Patent No 8 148 515 Materials from Biotium are sold for research use only and are not intended for food drug household or cosmetic use Page 2 of 3 Appendix AccuClear Assay Protocol for the AccuLite 470 Fluorometer Sample Preparation Note if using Mini Glass Tubes 100 uL sample volume can be used Scale all volumes in the reaction working solution and DNA proportionally 1 Prepare working solution as described in the AccuClear protocol 2 For each sample to be tested pipette 200 uL of the working solution into a 0 2 mL thin walled clear PCR tube To test samples in triplicate prepare three tubes for each sample Prepare two additional tubes for standards 3 Prepare standards Only the 0 ng DNA standard blank and 100 ng DNA standard are required Pipette 10 uL of AccuClear Buff
12. ttes and reagent reservoirs can be used to increase throughput Black plates are recommended to minimize fluorescence bleed through between wells We have found that black 96 well plates from Greiner Bio One or Corning give the most consistent signal to noise ratio at low DNA concentrations Continued next page Page 1 of 3 7 Add 10 uL of each dsDNA standard into its own separate well containing working solution and mix well by pipetting up and down 8 Pipette 10 uL of each unknown DNA into its own separate well containing working solution and mix well by pipetting up and down 9 Incubate the microplate at room temperature for 5 minutes in the dark 10 Measure fluorescence using a microplate reader to set to 468 nm excitation 507 nm emission maxima or other filter combination for detecting green fluorescence e g FITC filter set We recommend measuring fluorescence within 1 hour of performing the assay because fluorescence signal will decrease over time after DNA and dye are combined 15 decrease after 3 hours and 30 decrease after 6 hours 11 Generate a standard curve to determine the unknown DNA concentration see Figure 2 Average the triplicate values for each sample and subtract the average 0 ng DNA value from each data point Plot the fluorescence values for the DNA standards on the y axis and ng well DNA on the x axis and fit a trend line through these points to generate a standard curve with a y intercept 0 Use th
Download Pdf Manuals
Related Search