Qubit

The Qubit Fluorometer (Invitrogen 1.0 model) quantifies nucleic acid concentration with greater precision than the Nanodrop. The instrument is located in the BioMicro Center (BMC) and requires a kit (reagent + tubes) for diluting samples.

The protocol below describes the workflow for the dsDNA HS Assay Kit (Invitrogen Q32851).

Materials

• Your DNA sample, with concentration ranging from 5 pg/µL to 120 ng/µL

• Qubit dsDNA HS Reagent: light-sensitive orange solution, stored in a black bag in the drawer next to the genomics hood

• Qubit dsDNA HS Buffer: stored in bottle at room temperature in the genomics hood

• Qubit dsDNA HS Standards 1 and 2: tubes stored in Sven 4ºC door in Qubit box

• Qubit Assay Tubes (Invitrogen Q32856): special 0.5-mL tubes compatible with the instrument, stored in the cabinet next to the genomics hood

Prepare dilutions

1. Prepare the Qubit working solution: Dilute the Qubit dsDNA HS Reagent 1:200 in Qubit dsDNA HS Buffer. Prepare enough for 200 µL per sample plus 400 µL for the standards, e.g., for 8 samples, dilute 10 µL of reagent in 1.990 mL buffer (this includes enough extra to account for pipetting loss).

2. Prepare the standards: Dilute 10 µL each of Qubit dsDNA HS Standards 1 and 2 in 190 µL of working solution in the Qubit Assay Tubes, one tube per standard.

   Note

   When labeling the tubes, write on the cap, NOT on the side.

3. Prepare your samples: Dilute each of your DNA samples in working solution in the Qubit Assay Tubes. Choose an appropriate dilution factor based on your expected concentration. The Qubit dsDNA HS Assay is sensitive in the range 0.5-600 pg/µL (concentration of the diluted sample).

   • For samples 10-100 pg/µL, dilute 1:20 (i.e., 10 µL sample plus 190 µL working solution)

   • For samples 10-100 ng/µL, dilute 1:200 (i.e., 1 µL sample plus 199 µL working solution)

   • For samples with expected concentrations in between, choose any dilution between 1:200 and 1:20, inclusive

4. Vortex the diluted standards and samples. Incubate the samples at room temperature for 2 minutes. (This is as long as it takes to walk to the instrument in the BMC.)

Measure samples

1. Bring your samples to the BMC. The Qubit Fluorometer (Invitrogen 1.0 model) is in the back room on one of the lab benches.

2. Select the “Quant-IT dsDNA, HS” assay, then follow the prompts on the instrument to insert each standard and then your diluted samples, one at a time. Simply place the tube in the chamber (under the black lid, no need to push), close the lid, and press “Go” to measure each tube.

   

   Left: Choose the “dsDNA, HS” assay. Right: The instrument first prompts you to insert and measure Standard 1.

3. Record the measured concentration for each diluted sample. It will display in ng/mL, which is equivalent to pg/µL. Convert these values to the actual concentration of your samples based on the dilution factor.

4. When finished, insert the blank tube back into the chamber. You can dispose of the diluted samples and standards.