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plate set up - (Apr/09/2008 )

I'm a newbie of real-time PCR technique and I really need your advices!
I begun to prepare my reactions on my own, and however I'm confident about theory, I still feel quite "goofy" when I'm setting up the plate.
In other words, I would like to know how other people practically set up their plates.
Can you please suggest me tips and advices to follow when preparing dilutions, mixes, reactions, plate...and so on?

Thanks in advance!


The first plates can be a little confusing. My tricks not to get lost:

1. Open a new box of tips per plate, and use them in the same order as the plate you're loading, that way you always know which well are you in.
2. If you use white plates, you can get lost even when dispensing the mastermix. I dispense the mastermix with an electronic dispenser and set it up to 8 repeats, and dispense one column of the plate at a time.
3. I order my cDNA tubes in the same order they go in the plate, and move them a position once I've used them.
4. I dispense the cDNA in the wall of the well, that way i can see the drop in the wells that are already loaded.

It might look like a lot of things to do to make sure you are not getting lost, but believe me, this way I can set up the plates quickly and without having to think at all! Autopilot mode wink.gif

-erica arborea-

Also helpful is a dye in the mix. Sigma sells a red dyed master mix, containing cresol-red. You can add the cresol red yourself, and avoid paying their premium.


...but is cresol red compatible with every fluorophore?