I am going to use real time PCR using Taqman assay. I have experience of RT-PCR by using SYBR but I have no idea about Taqman. Can anyone suggest a protocol for Taqman?
- the use of a probe (for the quenching). You could use already made probes (commercial or from db) or define your own primers and probes using primer3.
- the enzyme/reaction mix, (not the mix of sybr green mix, no fluorescent molecule, not the same enzyme). I use an already made mix for TaqMan reaction.
- the wavelenght and the method of fluorescence detection (My qPCR system is a LC480).
The Master Mix will be different from SYBR green master mix. There are Taqman Master mix available.
There will be no need to do Melting Curve step and analysis, as you will have specific PCR product.
You must know which fluorescence dyes are used in your Probes and even you can do Multi-plex PCR in one reaction, if the probes and fluorescence are different.