Qubit4 Fluorometer Protocol: Invitrogen dsDNA HS Assay Kit (REF 32854)

Set up two assay tubes (Qubit assay tubes REF Q32856) for the standards (Standard #1 and Standard #2) and one assay tube for each sample.

Preparing working solution:

Dilute the Qubit reagent 1:200 in Qubit buffer: 1µL of *Qubit dsDNA HS Reagent 200X (multiplied by number of samples + one extra sample) for every 199µL of Qubit dsDNA HS Buffer (multiplied by number of samples + one extra sample). Make enough mix for the total number of samples including Standard #1 and Standard #2.

Prepare standards separately (Standard #1 and Standard #2): 190µL of working solution and 10µL of standard, to a final volume of 200µL for each standard.

Tip: Slowly mix up and down with pipette tip.

Prepare samples: 198µL of working solution and 2µL of extracted DNA, to a final volume of 200µL for each sample.

Tip: When pipetting DNA, make sure to mix slowly with pipette tip to prevent shearing. Slowly mix this mixture up and down with pipette tip as well.

Vortex all tubes and centrifuge briefly. Remove any bubbles before inserting tubes into the Qubit4 Fluorometer.

Setting up the Qubit4 Fluorometer: (a) select dsDNA, (b) choose dsDNA HS.

Insert standard tubes into the Qubit Fluorometer and take readings. Note: make sure that standard readings are accurate before moving on to the samples. (Standard #1: low, Standard #2: high). Specify the volume of DNA used and then take readings of your samples.