TITLE: DNA Quantification using the Qubit Fluorometer
AUTHORS: Julie Haendiges, Ruth Timme, Padmini Ramachandran, Maria Balkey

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This procedure outlines the protocol for quantitation of gDNA for subsequent WGS. </div><div class = "text-block">This document applies to all laboratory personnel in the Division of Microbiology (DM) as well as laboratories in the GenomeTrakr Network.</div><div class = "text-block"><span style = "font-weight:bold;">Complete in order:</span></div><div class = "text-block"><span style = "font-weight:bold;">1. DNA Extraction (Manual DNA Extraction </span><span style = "font-weight:bold;font-style:italic;">or</span><span style = "font-weight:bold;"> Automated DNA Extraction using the Qiacube)</span></div><div class = "text-block"><ul style = "list-style-type:disc;"><li style = "counter-reset:ol0;list-style-type:disc;">Step-by-step procedures to obtain high quality DNA from isolates in TSB for whole genome sequencing</li><li style = "counter-reset:ol0;list-style-type:disc;"></li><li style = "counter-reset:ol0;list-style-type:disc;"><span style = "font-weight:bold;">2. DNA Quantitation (included SOP)</span></li><li style = "counter-reset:ol0;list-style-type:disc;">Quantitation of extracted DNA using the Qubit Flourometer </li><li style = "counter-reset:ol0;list-style-type:disc;"></li><li style = "counter-reset:ol0;list-style-type:disc;"><span style = "font-weight:bold;">3. Library Preparation for WGS (Library preparation using Illumina DNA Prep </span><span style = "font-weight:bold;font-style:italic;">or </span><span style = "font-weight:bold;">Library Preparation using Illumina Nextera XT )</span></li><li style = "counter-reset:ol0;list-style-type:disc;"><span>Library preparation using NexteraXT </span><span style = "font-style:italic;">or </span><span>Illumina DNA Prep (previously Nextera DNA Flex)</span></li></ul></div><div class = "text-block"><span style = "font-weight:bold;">2. DNA Quantitation (included SOP)</span></div><div class = "text-block"><ul style = "list-style-type:disc;"><li style = "counter-reset:ol0;list-style-type:disc;">Quantitation of extracted DNA using the Qubit Flourometer </li><li style = "counter-reset:ol0;list-style-type:disc;"></li><li style = "counter-reset:ol0;list-style-type:disc;"><span style = "font-weight:bold;">3. Library Preparation for WGS (Library preparation using Illumina DNA Prep </span><span style = "font-weight:bold;font-style:italic;">or </span><span style = "font-weight:bold;">Library Preparation using Illumina Nextera XT )</span></li><li style = "counter-reset:ol0;list-style-type:disc;"><span>Library preparation using NexteraXT </span><span style = "font-style:italic;">or </span><span>Illumina DNA Prep (previously Nextera DNA Flex)</span></li></ul></div><div class = "text-block"><span style = "font-weight:bold;">3. Library Preparation for WGS (Library preparation using Illumina DNA Prep </span><span style = "font-weight:bold;font-style:italic;">or </span><span style = "font-weight:bold;">Library Preparation using Illumina Nextera XT )</span></div><div class = "text-block"><ul style = "list-style-type:disc;"></ul></div><div class = "text-block"><span style = "font-weight:bold;">4. Sequencing using Illumina MiSeq</span></div><div class = "text-block"><span style = "font-weight:bold;">5. Data Quality Checks and NCBI Submission</span></div></div>

[STEPS]
?. [Qubit Working Solution and Sample preparation]
Set up the required number of Qubit tubes for standards and samples. The Qubit dsDNA assays require two standards for calibration. Although, calibrations do not have to be performed each time it is recommended to perform prior toquantifying samples with critical input values.
?. [Qubit Working Solution and Sample preparation]
Label the tube lids. (Do not label the side of the tube as this could interfere with the sample reading), label tubes for standards as S1 and S2.
?. [Qubit Working Solution and Sample preparation]
Prepare the Qubit working solution by diluting the Reagent in a 1:200 ratio in Buffer. It is recommended to add an additional sample for overage.
?. [Qubit Working Solution and Sample preparation]
Add 190 μl of working solution to each of the standard tubes.
?. [Qubit Working Solution and Sample preparation]
Add 10 μl of each standard to the appropriate tube, then mix by vortexing.
?. [Qubit Working Solution and Sample preparation]
Add 198 μl of working solution to each sample tube.
?. [Qubit Working Solution and Sample preparation]
Add 2 μl of each sample to the appropriate tube, then mix by vortexing. The final volume should be 200 μl.
?. [Qubit Working Solution and Sample preparation]
Allow all tubes to incubate at room temperature for 2 minutes.
?. [Qubit Working Solution and Sample preparation]
Proceed to Reading Standards and Samples for the appropriate instrument.
?. [Reading Standards and Samples with the Qubit 3.0 Fluorometer]
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 3.0 Fluorometer]
If calibration is being performed, press Read Standards to proceed. (If a calibration is not necessary, you can omit the following steps)
?. [Reading Standards and Samples with the Qubit 3.0 Fluorometer]
Insert the tube containing Standard #1 into the sample chamber, close the lid, and press Read Standard. When reading is complete, remove the tube.
?. [Reading Standards and Samples with the Qubit 3.0 Fluorometer]
Insert the tube containing Standard #2 into the sample chamber, close the lid, then press Read Standard. When reading is complete, remove the tube.
?. [Reading Standards and Samples with the Qubit 3.0 Fluorometer]
The instrument displays the results of the calibration, for further information on interpretation of the results, refer to the Qubit 3.0 Fluorometer User Guide.
?. [Reading Standards and Samples with the Qubit 3.0 Fluorometer]
Press Read Samples to proceed
?. [Reading Standards and Samples with the Qubit 3.0 Fluorometer]
In the Sample Volume screen, select the volume of sample added to the tube and the output concentration units (i.e. ng/μl)
?. [Reading Standards and Samples with the Qubit 3.0 Fluorometer]
Insert the first sample tube into the sample chamber, close the lid, and then press Read Tube.
?. [Reading Standards and Samples with the Qubit 3.0 Fluorometer]
The software displays the results of the 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 readings and results recording for each additional sample.
?. [Reading Standards and Samples with the Qubit 4.0 Fluorometer]
The Qubit 4.0 has an on-board Reagent calculator that can be used to quickly determine the correct amount of reagent and buffer required to prepare the working solution.
?. [Reading Standards and Samples with the Qubit 4.0 Fluorometer]
On the Home screen, press Reagent Calculator
?. [Reading Standards and Samples with the Qubit 4.0 Fluorometer]
Enter the total number of samples and standards that you will be running.Optional: Select Include overage if you want to include an additional tube in the total calculated volume.
?. [Reading Standards and Samples with the Qubit 4.0 Fluorometer]
Press Done to return to the Home screen to run your Qubit Assay
?. [Reading Standards and Samples with the Qubit 4.0 Fluorometer]
On the Home screen, select dsDNA, then dsDNA: High Sensitivity or dsDNA:Broad Range depending on the kit being used.
?. [Reading Standards and Samples with the Qubit 4.0 Fluorometer]
If calibration is being performed, press Read Standards to proceed. (If a calibration is not necessary, you can omit the following steps)
?. [Reading Standards and Samples with the Qubit 4.0 Fluorometer]
Insert the tube containing Standard #1 into the sample chamber, close the lid, and press Read Standard. When reading is complete, remove the tube.
?. [Reading Standards and Samples with the Qubit 4.0 Fluorometer]
Insert the tube containing Standard #2 into the sample chamber, close the lid, then press Read Standard. When reading is complete, remove the tube.
?. [Reading Standards and Samples with the Qubit 4.0 Fluorometer]
The instrument displays the results of the calibration, 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 Fluorometer]
Press Read Samples to proceed.
?. [Reading Standards and Samples with the Qubit 4.0 Fluorometer]
In the Sample Volume screen, select the volume of sample added to the tube and the output concentration units (i.e. ng/μl)
?. [Reading Standards and Samples with the Qubit 4.0 Fluorometer]
Insert the first sample tube into the sample chamber, close the lid, and then press Read Tube.
?. [Reading Standards and Samples with the Qubit 4.0 Fluorometer]
The software displays the results of the 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 readings and results recording for each additional sample.
?. [Reference Documents]
Qubit 3.0 Fluorometer User GuideQubit 4.0 Fluorometer User GuideQuick Reference Qubit AssaysQubit dsDNA HS Assay GuideQubit dsDNA BR Assay Guide