Ask if the standards for your assay of choice have been run today. Ideally, the standards should be run every day the assay is used. However, it should not make that much difference if the standards are not run. Instructions involving standards will be in purple throughout this protocol.
Retrieve the standards (Standard 1 and Standard 2) from the Beiting Lab 4°C fridge and set them out on the bench for around 30 minutes.
Retrieve Qubit tubes, the correct Qubit buffer, and the box of Qubit dyes from the drawer labeled “Qubit Supplies”.
Qubit dyes are heat and light sensitive. They remain in the box unless you are actively aspirating dye.
Label Qubit tubes on the top of the tube.
Add buffer and dye (ensure that they match) in an Eppendorf tube in the correct ratio according to the number of reactions you need.
- If you are using standards: rxns = n + 2 + 1
- If you are not using standards: rxns = n + 1
Amount for 1 reaction (µL)
Vortex your Master Mix
Add 180-199 µL Master Mix to each tube receiving sample (usually 198 µL)
- Add 190 µL Master Mix to each tube receiving a standard
Add 1- 20 µL sample to the corresponding tube (usually 2 µL)
- Add 10 µL of each standard (remember, there are 2 for each assay) to the correct tube
Cap each tube tightly and vortex each for 5 seconds
Let the tubes sit at room temperature for 2 minutes
Bring the tubes to the Qubit
From the Home screen, press the correct type of nucleic acid
Choose “high sensitivity” or “broad range”
If you are running standards, choose “Read Standards”
- The Qubit will guide you through the steps for reading standards. Standard 2 should be at least 10X higher than Standard 1.
If you are not running standards, choose “Read Samples”
Indicate the correct amount of sample added to each tube
Read your samples and record the concentration
Samples in Qubit tubes are viable at room temperature for up to 3 hours