Diluting and pooling - last step

I have now prepared and amplified all my samples. I used 12 cycles and 8ul of product so that I wouldn't blow through the entire 20ul of library if something went wrong. Most of these had plenty of product, the couple that didn't I re-amplified for 14 cycles. For the dilution, I used the values from the nanodrop rather than the bioanalyzer as Sara has had some issues with pooling based on bioanalyzer values.

Here are the bioanalyzer traces of the completed pools:

The final concentration is a little low, but as the sequencing facility only needs ~10ul of 10nmol/L it should be fine. I actually ended up making 4 pools as the first two didn't stack up well on the qPCR. I think this was because I was pipetting such small volumes that the liquid was evaporating and changing my concentration. For the second set (Pools 3 and 4, seen above) I made sure that my sample volumes were never less than ~10ul. It seemed to work much better. Here's the qPCR trace of 5 of my samples. They should all be right on top of each other (except for the light blue one which is the no-template-control:

There is only about 1 cycle spread from the first one to the last, whereas the pools 1 and 2 had at least a 4 cycle spread. 

S~ is now setting up the account at Utah and I'll be sending the samples as soon as all the paperwork goes through. (We've had some issues with sequencing at Berkeley and so we're gonna try a new facility.)