Fluorsence Problem

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Kelly M.
Kelly M.'s picture
Fluorsence Problem

I'm trying to do a serial dilution of Alexa 488 in a 96 well plate to create a concentraion curve (hopefully).  I've done the experiment twice now, and the valuse I get from the plate reader are completely opposite from what we are expecting.  For example, my most concentraited sample gives the lowest readings and my least concentraited gives the highest.  My blank (PBS) is also giving a very high reading.  
 
To answer the obvious, I have the wavelength correctly set.  The plate is in the correct orientation. I've done my absolute best to minimize light exposure.  All dilutions are done in/with PBS.  We are using a black side, clear bottom 96 well plate with a volume of 200 uL.  We've read from the top and the bottom with no noticable difference in results.  We've changed the sensitivity on the scan with no real results.

Any thoughts on why I keep getting non-sensical values or how you'd go about creating a known curve for Alexa concentrations?