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 16 ul of Mastermix to each well needed.
Add 4 ul of each sample.
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”
Assign Standards Values:
Std1: 20, Std2: 2, Std3: 0.2, Std4: 0.02, Std5: 0.002, Std6: 0.0002
Navigate to “Instrument” tab and change cycle parameters to:
Temp C | Cycles | Time (m:ss) |
|---|---|---|
94 | 1X | 0:01 |
95 | 1X | 5:00 |
95 | 35X | 0:30* |
60 | 35X | 1:00* |
*These might change if we create any significantly longer products.
Hit the Save icon and ensure that it is being saved as an “.sds” document in the folder “C:/Applied Biosystems/SDS Documents”
Double Check Layout and Instrument Tabs Are Correct.
Press circular indent in the darker gray region to eject tray. Load Plate with A1 lined up with A1 mark.
Press “Start” in software. If Start is not an option, close “7500 System Software” and reopen. Reopen the file you just created and try “Start” again.
Always turn instrument off when finished for the day.