Cycle number in the TruSeq protocol

The qPCR results showed that 13-14 cycles would be excellent for my libraries (see this post). However, the amount of library that I started with was 1 40th of the total library (i.e. 0.5uL). If 0.5ul achieves the exponential phase in 13 cycles, then 8uL should achieve exponential phase in 9 cycles:
starting      cycles to
   ul         exponential
0.5              13
1                 12
2                 11
4                 10
8                  9
16                8

Thus I used 8uL of my libraries and amplified them for 9 cycles. This resulted in some libraries that were perfectly amplified and some that were not amplified enough. The top sample is great, a small even peak (and no secondary hump under the 1500 marker). The second sample does have a peak in the right area, but hasn't been amplified enough - there is not enough library there to sequence.

Fortunately, I only used 8uL of my pre-amplification library, not the entire 20uL just in case this issue arose. So now, instead of going back and re-preparing the entire library, I can just take and amplify the remaining parts. 

I'll need to talk to J~, as there are some serious batch effects (the first 8 all look like the top image, while the second 8 all look like the bottom image) but it looks like 8ul at 11 or 12 cycles will work fine for most everything without over-amplifying the library. As I will be comparing transcript levels, it is important that all the libraries are amplified the same number of cycles. 

Over-amplification is also bad because at that point in the PCR, the DNA molecules are being heated and cooled without being replicated. Thus they will fall apart and re-anneal repeatedly. They will often re-anneal with a DNA of a different sequence. This happens because the adapters have the same sequence, while the center sequences vary, two different molecules can meet eachother and the adapters on the ends will anneal while the middle region will for a big loop. This will mess up the sequencing machine and you'll get bad results. You know if you've over-amplified a library because there will be a low hump that around the 1500 marker. This is the weird hybridized molecules which travel slowly through a gel and so look quite large. In fact they are not any larger than the rest of the library and so can't be selectively removed via their size. The only thing that remains at that point is to re-amplify the library.

In short, I need my samples amplified more, but not too much more, so a cycle or two should be fine. J~ will be able to help me out with that.