actually got the thing to work (pretty damn well  ). however, will never do that again, will always use a premix, regardless of what my supervisor says.

I really wonder- those technicians at the companies can't be magicians as well.. there should be a way to figure out how to make a self-mixed solution. At the moment one complete plate run is about 100$ which is just ridiculous.
If you make a premix for all experiments there should not be a difference.

Here is what I tried so far (using SYBR green 10000x stock from molecular probes):

I tested different concentrations of SYBR, the final concentration of 100000x- 150000x worked best so far with all TAQs I used. Although I did not try higher concentrations with more Mg+, as someone suggested in another forum.

My biggest problem is that I get background melting-curves at lower cDNA concentrations, it might be primer dimers or contamination, but I never see this with commercial mix. I tried both an inhibited (Eppendorf Hotmaster) and normal (Qiagen) TAQ.

Some companies include dUTPs (for PCR (not RT-PCR)) - does anyone know what the advantage of this is?

Also - does anyone know some more "tricks"?


C

The problem I've encoutered with mixing my own is that although I may get acceptable results, if I were to repeat the experiment a week later, they would be different. From talking to the Bio-Rad reps, they include "stabilizers" for the sybr green which I assume works since our melt curves have been beautiful. The premixed kits just seem a lot more reproducible.

Greg

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