The KAPA Library Quant Kit (Illumina) that we use has a limited range of detectability. Too high leads to signal saturation and too low leads to not enough signal. The standards range from 20 pM to 0.0002 pM. Our library prep methods produce products in the 1-100 nM range. Generally, all samples must be diluted 1 to 1000.
Add 1 ul sample to 999 ul ultra pure water. Vortex and quick centrifuge sample dilutions.
Generally, all samples, standards, and ntcs should be done in triplicate. Enter the appropriate number into the top “# of rxns” cell below.
Keep MasterMix in the dark as much as possible. The qPCR dyes are light sensitive and degrade with exposure.
Add MasterMix and Samples:
Add 16 ul of Mastermix to each well needed.
Add 4 ul of each sample.
Setup Instrument For Run
Open “7500 System Software”
Hit New File Icon
Name Plate Appropriately (Copy from Confluence Page in Question). Hit “Next”.
Select “Unknown”. Hit “Add”. Click “Next”
Highlight All Wells To Be Used And Assign “Unknown” Detector
Assign All Wells Their Task From The Dropdown next to “Unknown” detector. Choices Are “NTC”, “Unknown”, or “Standard”. Click “Finish”