A master mix is the reaction minus the template and the primers, sold as a concentrate so that one pipetting step replaces five. That is its real value: the largest single source of variance in a plate is the operator, and a mix removes most of the chances to introduce it. What the tube contains, and whether the enzyme in it suits the chemistry you are running, is where the choices actually are.
What a 2x PCR master mix contains, and why 2x is the format
The concentrate holds the polymerase, the nucleotides, magnesium and the buffer, and in a dye-based mix the intercalating dye as well. It is supplied at twice the final strength because that leaves half the reaction volume for the template and primers, which is the widest useful split: a higher concentrate leaves too little room to dilute a template, and a lower one leaves too little room for the mix. Anything the vendor adds beyond that list, a passive reference dye or a uracil glycosylase, is a formulation decision you should know about before a protocol depends on it.
Reverse transcriptase PCR master mix, and when an RT-PCR master mix should be two tubes
A one-step formulation puts the reverse transcriptase and the polymerase in the same tube and runs both reactions without opening it, which is the right choice when contamination is the main risk and the target is fixed. A two-step reaction transcribes first, then takes an aliquot of the resulting complementary DNA into a separate amplification, and that is the right choice when the same sample must be assayed for several targets or when the transcription needs its own conditions. The one-step tube is a compromise by construction, because the two enzymes do not share an optimum.
LAMP master mix, and why isothermal is a different chemistry
Loop-mediated amplification runs at one temperature with a strand-displacing polymerase, so its mix is not a PCR mix with a different label: the enzyme, the buffer strength and the magnesium are all different, and a thermal cycler is not required. It is also far more sensitive to primer design than to the mix, which is worth knowing before a failed reaction is blamed on the reagent. Treat it as a separate method that happens to amplify DNA, not as a faster version of what you already run.
Setting up a master mix PCR run, and where variance creeps in
Most of the spread in a plate is added after the mix is chosen. Thaw once and keep aliquots, because repeated freezing degrades the nucleotides before it touches the enzyme. Make enough for the plate plus a margin and dispense it before the template, so every well sees the same batch. Mix the concentrate gently and completely, since the dense components settle and an unmixed tube gives a gradient across the plate that looks exactly like a pipetting error. None of this is about the product; it is about not undoing what the product was bought to fix.
Questions people ask about qpcr master mix
What does PCR master mix 2x on the label mean?
That the concentrate is twice the final reaction strength, so you add an equal volume of template and primers to reach 1x. Labels write it in either order, and it says nothing about what is in the tube.
Which RT PCR reagents does a one-step kit already include?
The reverse transcriptase, the polymerase, the nucleotides, magnesium and the buffer. You supply the template and primers. It does not include a separate transcription buffer, which is one of the compromises of putting both enzymes in one tube.
Can I use a qPCR mix for ordinary endpoint PCR?
Usually yes, and the dye is then simply along for the ride. The reverse is not safe: an endpoint mix has no dye and often no passive reference, so it will amplify without reporting anything a real-time instrument can read.