Testing the shot volume

So since my lasy post I have been varying the volume of the shot driven through the machine. I'm pushing Flouroscein against Flouroscein so that no reaction is observed but it will show a change in the florescence as the temperature changes. Before I was using a machine with a 60 ul cell, however this broke so I am now using one with a 5 ul cell. The advantage of this is that it is better suited for cold temperatures and I can use smaller shot volumes. The volumes I used where 20, 30, 40, 50, 60, 80, and 90 ul. However the traces of the florescence that I obtained only showed any temperature change at 80 and 90 ul, this would suggest that the device on the cell is cooling down or heating up the sample instantly from 20 - 60 ul. The way in which I can determine this is that if there is no temperature change then the trace will come out flat, if there is a heating up effect then the florescence will shoot up then return to the starting amplitude, if there is cooling then there will be a drop followed by a rise in amplitude. This was fairly dull as most of the time I was looking at traces of just horizontal lines, hopefully the rest of the week will be better as I am moving on to finding the ADP dissociation constant when mixed with S1, actin and ATP.