you need SYBR (or similar) to detect real-time results (unless you are using a probe-based system, but the link you sent is not).

Add to your master mix; it's typically a 10,000 fold dilution.

It might be worth looking into ROX to help tighten up your curves. (helps normalize for pipetting errors).

You have to have a fluorescent reporter added to your PCR mix to use the qPCR machine. You can add it manually, but I'd recommend just buying the pre-made master mix. I would suggest you talk with a colleague about your experiment before you continue. It sounds like you are very new to qPCR and you should know what your data will look like and how to do the analysis before you start setting up reactions. Good luck!

Hi Hannah,

I'm totally agree with Katie and Matthew also. You need a fluorescent dye (e. g. SYBR Green - you can add it separetly to your master mix) in QPCR measurements. Your chemistry is primer based only with intercalating dyes. Many user use only 1 normalization gene. But in many systems hasn't work well. So you need to test what will be the most stable expression normalization gene in you system. You shoud use min. 3-6 housekeeping genes to adequatley normalize your GOI expression. In 1st test I would try Actb, Gapd, Rpl13a genes.

You definitely should use NTC (No Template Control) and NAC (No Amplification Colntrol) beside your housekeeping genes with every master mix group.

Because of you run a LC96, you don't need to use passive reference dye (ROX). The LC96 hasn't calculate its fluorescence.

If you need help in QPCR experiments, just feel free to write. I try to answer!

Bests,

Tamas