Our machine not sure of the specific type just now, gives both temperatures in the output as if it were subsequent cycles. So if you set 40 cycles you get 80 cycles raw output with the two temperature alternating. Does that make sense? I then use these raw fluorescence values to calculate ct values. Does that help.

Dear Morgan,

Here is our adaptation for the Cawthon assay for  CFX96. We do use separate plates and gene study:

https://www.researchgate.net/publication/236192892_An_in_vitro_expansion_score_for_tissue-engineering_applications_with_human_bone_marrow-derived_mesenchymal_stem_cells

Article An in vitro expansion score for tissue-engineering applicati...

I'm only getting one Cq and one fluorescence value per cycle per well.. I ran the reaction for 40 cycles and am getting 40 values. The protocol is set up to take two reads - still can't figure out how to get both read values...

If you export the data does it then also only give you the 40 measurements?

Yes, when I export the data I only get 40 values... I'm going to speak with the BioRad representative on Tuesday. Hopefully he can help.

Hi Morgan.. I had a little google and it should be possible on your machine, because these guys also did it using monochrome multiplex qpcr: http://www.ncbi.nlm.nih.gov/pubmed/21974787

Probably you do need to fiddle around with the software a bit. 

If you only get 40 values that is also not correct you need to export raw fluorescence reads (you need 96*40 reads) or is that what you mean? ROX & SYBR.

Let us know what the solution is.

Solution:  you need to tell the BioRad CFX software which "step" you're on in the "quantitation" window - there's a box to select reaction step below the standard curve window. Since the run included two reads, one at step 7 (low-temp read) and one at step 9 (high temp read), you can change between these steps to get the early and later Cq values.  Then, you can output these data separately.

Great!