TITLE: C-SOP-201: Genomic DNA Quantification using a Qubit Fluorometer
AUTHORS: Mihir Kekre

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><div class = "justify" style = "text-align:left">In order to prepare high quality double-stranded DNA libraries for Illumina WGS, extracted genomic DNA needs to be accurately quantified. Qubit Fluorometers detect fluorescent dyes that are specific to the target of interest. These fluorescent dyes emit only when bound to specific target molecules, even at low concentrations. This provides obvious advantages over UV absorbance quantitation, which measures anything absorbing at 260 nm — DNA, RNA, protein, free nucleotides, or excess salts. Moreover, UV spectrophotometry often does not have the sensitivity to accurately measure low concentrations of DNA and RNA. The Qubit dsDNA broad-range (BR) and high-sensitivity (HS) assays are highly selective for double-stranded DNA (dsDNA) over RNA and single-stranded DNA (ssDNA). </div></div><div class = "text-block"><div class = "justify" style = "text-align:left"></div></div><div class = "text-block"><div class = "justify" style = "text-align:left">This protocol has been adapted from dsDNA quantification assays developed by ThermoFisher Scientific.</div></div><div class = "text-block"><div class = "justify" style = "text-align:left"></div></div><div class = "text-block"><div class = "justify" style = "text-align:left">Variations of this method include quantification assays from Promega:</div></div><div class = "text-block"><div class = "justify" style = "text-align:left"><a href="https://www.protocols.io/view/dna-quantification-using-the-quantus-fluorometer-7pzhmp6" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">DNA quantification using the Quantus fluorometer</span></a></div></div><div class = "text-block"><div class = "justify" style = "text-align:left"></div></div><div class = "text-block"><div class = "justify" style = "text-align:left"></div></div><div class = "text-block"><span>This is an open-access protocol distributed under the terms of the </span><a href="https://creativecommons.org/licenses/by-nc-sa/4.0/legalcode" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">Creative Commons Attribution-NonCommercial-ShareAlike</span></a><span style = ":;">.</span></div><div style = "text-align :; float : ;"><img style = "" src = "https://protocols-files.s3.amazonaws.com/files/cuzrbf8zp.png?X-Amz-Content-Sha256=UNSIGNED-PAYLOAD&X-Amz-Algorithm=AWS4-HMAC-SHA256&X-Amz-Credential=AKIAJYFAX46LHRVQMGOA%2F20210115%2Fus-east-1%2Fs3%2Faws4_request&X-Amz-Date=20210115T144235Z&X-Amz-SignedHeaders=host&X-Amz-Expires=604800&X-Amz-Signature=4d30786ca7aacf846ebb45f629340425a877a4d64c2540df606a3635695c861e" /></div><div class = "text-block"><div class = "justify" style = "text-align:left"></div></div><div class = "text-block"><div class = "justify" style = "text-align:left"></div></div></div>

[STEPS]
?. [Before starting]
The buffer and BR/HS reagent (dye) should be stored at  while the standards must be stored at . The reagent is light and humidity sensitive, it should be stored in the dark and exposed minimally during assay and sample preparation. When stored as directed, the reagent has a shelf-life of about 6 months.
4 Room temperature
4 °C
?. [Qubit Mastermix and Standard/Sample Preparation]
Set up the required number of Qubit tubes for standards and samples.
i. The Qubit dsDNA assays require two standards (#1 and #2) for calibration.ii. If you have already performed a calibration on the Qubit machine for the selected assay you can use the previous calibration stored on the machine. It is recommended to perform a new calibration for every sample batch but a same-day calibration would be fine to use for multiple batches.
?. [Qubit Mastermix and Standard/Sample Preparation]
Label the sample assay tube lids with sample IDs and the standard tubes lids as S1 and S2.
i. Use only thin-wall, clear, 0.5mL PCR tubes. The recommended consumables are listed under Materials (Cat. No. Q32856).ii. Do not label the side of the tube as this could interfere with the sample reading
?. [Qubit Mastermix and Standard/Sample Preparation]
Prepare a Qubit Mastermix for each sample and standard by diluting the Reagent in a 1:200 ratio with Buffer.Per sample: Qubit® dsDNA BR/HS Reagent: Qubit® dsDNA BR/HS Buffer:     To avoid pipetting errors, it is recommended to add some overage as indicated below in Table 1.Volume per sample (µL)For 10 samples (x 11)For 20 samples (x 21)For 30 samples (x 31)For N samplesQubit Reagent1112131N+1Qubit Buffer199218941796169199 x (N+1)
1 µl
199 µl
Volume per sample (µL)For 10 samples (x 11)For 20 samples (x 21)For 30 samples (x 31)For N samplesQubit Reagent1112131N+1Qubit Buffer199218941796169199 x (N+1)
To avoid any cross-contamination, it is recommended that the total amount of working solution required for samples and standards is removed from the working solution bottle and then added to the mastermix container instead of pipetting directly from the stock bottle to each tube.
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?. [Qubit Mastermix and Standard/Sample Preparation]
Aliquot the appropriate volume of Qubit Mastermix into each tube: Standard tubes: Sample tubes Next, to the appropriate tube:Add  of standards (S1 and S2).Add  of each sample.The final volume in each tube must be  once the samples/standards are added.
190 µl
198 µl
10 µl
2 µl
200 µl
i. The Qubit dsDNA assays require 2 standards for every round of calibration.ii. For samples that are suspected to have low yields of DNA, the sample tube volume can be decreased to anywhere between 180-198µL in order to supplement the final volume with a greater quantity of input DNA.iii. When adding 1–2µL of sample input, use a P-2 pipette for best results.
?. [Qubit Mastermix and Standard/Sample Preparation]
Cap each tube and then mix vigorously by vortexing for .Allow all tubes to incubate at  for .
0 Room temperature
?. [Reading Standards and Samples with the Qubit 4.0]
On the home screen of the Qubit, press dsDNA, then select dsDNA: High Sensitivity or dsDNA: Broad Range depending on the kit being used.
?. [Reading Standards and Samples with the Qubit 4.0]
INSTRUMENT CALIBRATION:If calibration is being performed, press Read Standards to proceed. (If a calibration is not necessary, you can omit steps 8.1 to 8.3).
?. [Reading Standards and Samples with the Qubit 4.0]
Insert the tube containing Standard #1 into the sample chamber, close the lid, and press Read Standard. When reading is complete, remove the tube and place back in the rack.
?. [Reading Standards and Samples with the Qubit 4.0]
Next, press Read Samples to proceed to the assay.
?. [Reading Standards and Samples with the Qubit 4.0]
Insert the tube containing Standard #2 into the sample chamber, close the lid, then press Read Standard. When reading is complete, remove the tube and place back in the rack.
?. [Reading Standards and Samples with the Qubit 4.0]
The instrument displays the results of the calibration immediately after S1 and S2 have been read. For further information on interpretation of the results, refer to the Qubit 4.0 Fluorometer User Guide.
?. [Reading Standards and Samples with the Qubit 4.0]
i. The software displays the results of each sample. The top value (in large font) is the concentration of the original sample. The bottom value is the dilution concentration. Record the concentration of the original sample, remove the tube and repeat steps 9.1 and 9.2 to record results for each additional sample.ii. To manage and import assay readings directly from the instrument, refer to the relevant Fluorometer User Guide in the next section.
?. [Additional Information & Troubleshooting]
For more information, refer to the following links depending on the model of instrument and type of assay being used.Quick Reference Qubit AssaysQubit dsDNA BR Assay GuideQubit dsDNA HS Assay GuideQubit 3.0 Fluorometer User GuideQubit 4.0 Fluorometer User GuideQubit Flex Fluorometer User Guide
?. [Reading Standards and Samples with the Qubit 4.0]
On the Sample Volume screen, select the volume of sample added to the tube and the output concentration units (i.e. ng/μl or other).
?. [Reading Standards and Samples with the Qubit 4.0]
Insert the first sample tube into the sample chamber, close the lid, and then press Read Tube.
?. [Before starting]
Prior to initiating the protocol, ensure that all active workbenches are cleaned with 80% ethanol, all relevant personal protective clothing is worn and the work area is prepared for DNA quantification according to local GLP guidelines.Create an organised bench space by clearing away all clutter in order to maximize work efficiency.