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

[DESCRIPTION]
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). 

This protocol has been adapted from dsDNA quantification assays developed by ThermoFisher Scientific.

Variations of this method include quantification assays from Promega:
DNA quantification using the Quantus fluorometer


This is an open-access protocol distributed under the terms of the Creative Commons Attribution-NonCommercial-ShareAlike.
  





[GUIDELINES]
Storage (Qubit BR/HR kit):
When not in use, the buffer and BR/HS reagent (dye) should be stored at 4 Room temperature while the standards must be stored at 4 °C. 
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.

[STEPS]
SECTION: Before starting
1. The buffer and BR/HS reagent (dye) should be stored at 4 Room temperature while the standards must be stored at 4 °C. 

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.
SECTION: Qubit Mastermix and Standard/Sample Preparation
3. Set up the required number of Qubit tubes for standards and samples. 

 

SECTION: Qubit Mastermix and Standard/Sample Preparation
4. Label the sample assay tube lids with sample IDs and the standard tubes lids as S1 and S2.
 

SECTION: Qubit Mastermix and Standard/Sample Preparation
5. 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: 1 µL
Qubit® dsDNA BR/HS Buffer:     199 µL

To avoid pipetting errors, it is recommended to add some overage as indicated below in Table 1.

 

 

SECTION: Qubit Mastermix and Standard/Sample Preparation
6. Aliquot the appropriate volume of Qubit Mastermix into each tube: 
Standard tubes: 190 µL
Sample tubes 198 µL


Next, to the appropriate tube:
Add 10 µL of standards (S1 and S2).
Add 2 µL of each sample.


The final volume in each tube must be 200 µL once the samples/standards are added. 
 



SECTION: Qubit Mastermix and Standard/Sample Preparation
7. Cap each tube and then mix vigorously by vortexing for 5 s.

Allow all tubes to incubate at Room temperature for 2 min.

SECTION: Reading Standards and Samples with the Qubit 4.0
8. On the home screen of the Qubit, press dsDNA, then select dsDNA: High Sensitivity or dsDNA: Broad Range depending on the kit being used.


SECTION: Reading Standards and Samples with the Qubit 4.0
9. 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).




SECTION: Reading Standards and Samples with the Qubit 4.0
9.1. 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.
SECTION: Reading Standards and Samples with the Qubit 4.0
10. Next, press Read Samples to proceed to the assay.


SECTION: Reading Standards and Samples with the Qubit 4.0
9.2. 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.
SECTION: Reading Standards and Samples with the Qubit 4.0
9.3. 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.
SECTION: Reading Standards and Samples with the Qubit 4.0
11. 
 

SECTION: Additional Information &amp; Troubleshooting
12. For more information, refer to the following links depending on the model of instrument and type of assay being used.

Quick Reference Qubit Assays

Qubit dsDNA BR Assay Guide

Qubit dsDNA HS Assay Guide

Qubit 3.0 Fluorometer User Guide

Qubit 4.0 Fluorometer User Guide

Qubit Flex Fluorometer User Guide
SECTION: Reading Standards and Samples with the Qubit 4.0
10.1. 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).
SECTION: Reading Standards and Samples with the Qubit 4.0
10.2. Insert the first sample tube into the sample chamber, close the lid, and then press Read Tube.
SECTION: Before starting
2. 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.