Aug 15, 2020

Public workspaceDNA Quantification using the Qubit Fluorometer

DOI
dx.doi.org/10.17504/protocols.io.bi8dkhs6
  • 1US Food and Drug Administration
  • GenomeTrakr
    Tech. support email: genomeTrakr@fda.hhs.gov
Julie Haendiges
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DOI: dx.doi.org/10.17504/protocols.io.bi8dkhs6
Protocol Citation: Julie Haendiges, Ruth Timme, Padmini Ramachandran, Maria Balkey 2020. DNA Quantification using the Qubit Fluorometer. protocols.io https://dx.doi.org/10.17504/protocols.io.bi8dkhs6
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License,  which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: July 31, 2020
Last Modified: November 11, 2021
Protocol Integer ID: 39909
Keywords: DNA Quantitation, Qubit dsDNA, Whole Genome Sequencing, GenomeTrakr,
Disclaimer
Please note that this protocol is public domain, which supersedes the CC-BY license default used by protocols.io.
Abstract
This procedure outlines the protocol for quantitation of gDNA for subsequent WGS.

This document applies to all laboratory personnel in the Division of Microbiology (DM) as well as laboratories in the GenomeTrakr Network.


Complete in order:
1. DNA Extraction (Manual DNA Extraction or Automated DNA Extraction using the Qiacube)
  • Step-by-step procedures to obtain high quality DNA from isolates in TSB for whole genome sequencing

2. DNA Quantitation (included SOP)
  • Quantitation of extracted DNA using the Qubit Flourometer

3. Library Preparation for WGS (Library preparation using Illumina DNA Prep or Library Preparation using Illumina Nextera XT )
  • Library preparation using NexteraXT or Illumina DNA Prep (previously Nextera DNA Flex)

4. Sequencing using Illumina MiSeq

5. Data Quality Checks and NCBI Submission
Guidelines
Abbreviations:
BR: Broad Range
DM: Division of Microbiology
HS: High Sensitivity
PI: Principal Investigator
ST1: Standard 1
ST2: Standard 2
Materials
MATERIALS
ReagentQubit dsDNA HS Assay KitThermo Fisher ScientificCatalog #Q32851
ReagentQubit assay tubesThermo Fisher ScientificCatalog #Q32856
ReagentQubit™ dsDNA BR Assay KitThermo FisherCatalog #Q32853
Supplies:
  • Pipette tips, sterile, filtered (assorted volumes)
  • 5ml round-bottom polystyrene tubes (Fisher Sci Cat# 14-959-2A or equivalent)

Equipment:
  • Qubit 3.0 or 4.0 Fluorometer
  • Vortex
  • Micropipettes, various volumes
  • Microcentrifuge


Safety warnings
Take proper precautions and wear appropriate PPE when handling potentially hazardous chemicals. Ensure that chemicals, spent containers, and unused contents are disposed of in accordance with governmental safety standards.

Qubit dsDNA High Sensitivity and Broad Range Reagent Kit: See ThermoFisher Scientifics SDSs for additional information
• HS/BR Reagent 200x in DMSO: GHS Category 4 for flammability, no data is available on the mutagenicity or toxicity of the reagent, it is known to bind nucleic acid so the same safety precautions should be used as when handling other potential mutagens.
• HS/BR Buffer: No GHS classification but may cause irritation with susceptible persons.
Before start
The buffer and reagent should be stored at room temperature while the standards must be stored at 4oC. The reagent is light and humidity sensitive, it should be stored in the dark and kept in the Ziploc bag with the desiccate pack. When stored as directed, kits are stable for 6 months.
Qubit Working Solution and Sample preparation
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 to
quantifying samples with critical input values.
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.
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.




Add 190 μl of working solution to each of the standard tubes.
Add 10 μl of each standard to the appropriate tube, then mix by vortexing.
Add 198 μl of working solution to each sample tube.
Add 2 μl of each sample to the appropriate tube, then mix by vortexing. The final volume should be 200 μl.
Allow all tubes to incubate at room temperature for 2 minutes.
Proceed to Reading Standards and Samples for the appropriate instrument.
Reading Standards and Samples with the Qubit 3.0 Fluorometer
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.
If calibration is being performed, press Read Standards to proceed. (If a calibration is not necessary, you can omit the following steps)
Insert the tube containing Standard #1 into the sample chamber, close the lid, and press Read Standard. When reading is complete, remove the tube.
Insert the tube containing Standard #2 into the sample chamber, close the lid, then press Read Standard. When reading is complete, remove the tube.
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.
Press Read Samples to proceed
In the Sample Volume screen, select the volume of sample added to the tube and the output concentration units (i.e. ng/μl)

Insert the first sample tube into the sample chamber, close the lid, and then press Read Tube.
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
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.
On the Home screen, press Reagent Calculator
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.
Press Done to return to the Home screen to run your Qubit Assay
On the Home screen, select dsDNA, then dsDNA: High Sensitivity or dsDNA:Broad Range depending on the kit being used.
If calibration is being performed, press Read Standards to proceed. (If a calibration is not necessary, you can omit the following steps)
Insert the tube containing Standard #1 into the sample chamber, close the lid, and press Read Standard. When reading is complete, remove the tube.
Insert the tube containing Standard #2 into the sample chamber, close the lid, then press Read Standard. When reading is complete, remove the tube.
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.
Press Read Samples to proceed.
In the Sample Volume screen, select the volume of sample added to the tube and the output concentration units (i.e. ng/μl)
Insert the first sample tube into the sample chamber, close the lid, and then press Read Tube.
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
Reference Documents