Note: This procedure assumes gDNA concentration in the range of 0-10ng/μl.
If samples cover a different range of concentrations, the procedure should be modified accordingly. We recommend a two step-dilution for samples with a broad range of concentrations.
- Create the standards.
You will need to run new standards for each batch of SYBR Green I working solution you use. You will need at least 10μL of each standard.
Use the standards from the Qubit HS DNA kit. The standards have a concentration of 10 ng/μL and 0 ng/μL. Prepare 12 uL of the standards as outlined in the following table:
|Standard (ng/μL)||Vol. 10 ng/μL Standard||Vol. 0 ng/μL Standard||Vol. 1 ng/μL Standard|
|10||12 μL||0 μL|| |
|7.5||9 μL||3 μL|| |
|5||6 μL||6 μL|| |
|2.5||3 μL||9 μL|| |
|1||2.4 μL||21.6 μL|| |
|0.5|| ||6 μL||6 μL|
|0.25|| ||9 μL||3 μL|
|0||0 μL||12 μL|| |
*NOTE- You can make a larger quantity of each standard and store them at 4 C.***NOTE- It is possible to use the 100 ng/μL Standard from the Qubit BR DNA Kit to increase the range of your standards, but be aware on the Tecan Plate Reader using SYBR Green I the fluorescence signal maxes out at around 20 ng/μL.
- Mix 5μl of concentrated SYBR Green I with approximately 25mL of TE in a clean reservoir to make a working dye solution.
- The same dye solution should be used across all samples and standards.
- Seal, vortex and centrifuge gDNA (~200rcf for 30s).
- Add 10 μl of gDNA to each well of a fluorometry plate.
- Recommended: 96-well pipette or multi-channel pipette.
- In 8 wells of fluorometry plate, add 10 μl of each DNA standard.
- Little plate-to-plate variability is seen, as long as the same dye solution is used.
- Dispense 190 μl of dye solution into each well.
- Incubate the DNA and dye in the dark for 5 minutes, e.g., put in a dark drawer or covered with foil.
- Read fluorescence on the plate reader using SYBR Green or GFP filters (e.g. Ex:470/40 and Em:520LP, top read). NOTE- This method should be in the Gresham Lab methods folder on the Tecan plate reader computer.
- If standard curve is not linear, allow the plate to sit longer and repeat.
- If the samples are outside the linear range of standards, repeat with a different dilution of sample.
- Calculate DNA concentration of each sample using by fitting a linear model to the standards, then calculate the volume of water needed to dilute each sample to the desired concentration (eg. for the Nextera Protocol this will be between 2-2.5 ng/μl.
- You can use the excel file associated with this page to input your standards and your DNA readings to calculate your concentrations.
- (Optional) Standardized gDNA libraries can be stored overnight at -20°C.
SYBR_Green_DNA_Conc_Calculation Sheet (Updated 07/14/2017 - Nathan)