Protocol #04
Amplicon quantification
Using QuantiFLuor dsDNA system from Promega on Qubit or Roche LightCycler 480II
Prep:
 01 Thaw the QuantiFluor dye. Quick vortex and quick spin. Keep it in the dark.
 02 Quick vortex and quick spin the Standard DNA.
 03 Prepare standards for quantification from the concentrated DNA standard.
You will need just 2 µL per standard. Don’t waste it. Use a two-fold serial dilution.
100 ng/µL → 50 ng/µL → 25 ng/µL → 12.5 ng/µL → 6.25 ng/µL → 3.125 ng/µL → 0 ng/µL
 04 Calculate how much of the working solution you need for your samples. Don’t forget
to include the five standards.
 For Qubit, you need 200 µL total per sample or standard.
 For LightCycler, you need 50 µL total per sample or standard.
 05 Prepare the working solution – QuantiFluor Dye 1:400 in 1× TE buffer.
e.g. To measure 10 samples and 5 standards on LightCycler, you will need 15×50=750 µL
total, meaning you need 2 µL of the QF Dye, 40 µL of TE and 758 µL of water.
 06 Mix the working solution properly. Vortex and quick spin.
 07 Combine the working solution and samples/standards.
 For Qubit, you need 198 µL of working solution and 2 µL of sample/standard.
You must use the optical grade 0.5 mL tubes.
 For LightCycler, you need 48 µL working solution and 2 µL of sample/standard.
You must use white plates or white strips and optical grade foils or caps.
 08 Vortex the samples. Do a quick spin.
 09 Incubate at RT for 3 minutes. Should be kept in dark.
 10 Measure the samples and standards. Write down the relative fluorescence.
 11 Construct the calibration curve in Excel and calculate the concentration of samples.