Why not optimise on the real time machine? - (Feb/08/2006 )

Can anyone give me a reason why most people optimise on a PCR before they move on to a real time? Is it just the expense and the probes?

I have been wondering because it seems that in my situation it makes more sense to optimise primer concentration, MgCl etc on the real time, since its faster, more sensitive, and you don't need to run a gel.

But maybe I've missed something?

I don't know how others approach this. I did a run of 'straight PCR' once to make sure that the primers would give product of the expected size, and not a bunch of primer-dimer

then, all my optimization as far as specific reaction conditions was done on the machine.

how do / did others set it up?

Hi - thanks for that.

Sounds like I am doing this similarly to you.

I had the impression that this is not the most common approach but maybe I'm wrong?

I use TaqMan probes, and I have only done optimization on real-time PCR...

I don't think anything else would accomplish exactly what I want - but I am fairly new at this so I don't think my opinion means much!!

I have no idea how others do it; I didn't have anyone show me. I reasoned that you could spend (waste!) a bunch of time optimizing straight PCR and have to change your conditions when you get to the machine, and that in the long run it would be more expensive

Yeah I agree! The real-time system can be so finicky that it is hard to imagine that conditions optimized for anything BUT it would work. And even those don't always work when you get new reagents, and you have to optimize again!