Help please- strange (and different) bioanalyzer results

[jlynn]

Hello Seq-answers community,

I need help interpreting bioanalyzer results and the background is below:

I have recently completed library prep using NEB next ultra ii DNA library kit for my ChIP-DNA samples. I made a mistake when diluting the adapters because I diluted them to my lowest concentrated sample and added the diluted adapters to all of my 6 samples even though some of them were at higher concentration and should have had 1/10 dilution of adapters rather than 1/25 (for low conc.). After figuring out why I had no DNA at the end of the lib prep, I was able to extract and clean up the DNA bound to the beads and from the supernatant containing size selected fragments that were not expected to be within the correct range. I then used the salvaged DNA to do another library prep, from end repair to the amplification of size selected fragments, hoping they were the correct size and had proper adapters this time. I quantified after amplification and had less yield than expected (<100ng) for all samples. Here is where I am confused and need some interpretations... I used the bioanalyzer with the HS DNA assay and got back the following electropherogram results. My input libraries seem ok and I think that the libraries are very diluted which is why it is a small peak. The other samples, my ChIP-DNA libraries, produced strange bioanalyzer readings. Please help me interpret these results.

[microgirl123]

To start with your ladder looks bad - your ladder peaks should be as high as or higher than your marker peaks at the ends. Does your ladder always look like that? If so, I'd contact Agilent and if not, I'd run another chip. Either way, I wouldn't start worrying about the samples until you sort out the ladder.

[jlynn]

Actually, since this is a unit in our shared core facility, for some reason the ladder was pretty much empty (<1ul). I had already set the chip up and then opened the ladder and said oh ****! So I added 5ul of 0.1% TE, vortexed, and loaded 1ul, which should explain the weak ladder. Do you think that it is worth doing another chip? Maybe with a less diluted library sample?