A week or so ago someone forgot to close the door on our enzyme freezer tightly.  I had just ordered $1,000 worth of NEB enzymes to make high throughput sequencing libraries too...  Before the meltdown, I made a couple of test libraries to be sure that the protocol was worked out.

One of my test libraries with a perfect library smear.  We extract the DNA in between the 200 and 300bp bands for sequencing.

I was really worried about the enzymes being thawed overnight, but I was told by a couple of different people that they'd be fine.  So I went ahead and processed all three of my experimental timpoints (probably a huge mistake...), and each timepoint has 6 different libraries (one for each DNA associated protein I yanked out of the cells).  So there were a total of 18 samples.  Problem is they all came out looking like this.

Notice how the smears aren't as smeary? The band you're seeing is called a primer-dimer.  It shows up when primers stick to one another and amplify eachother instead of the intended DNA target.  This probably means that the PCR adapters weren't added to my libraries :(

Now I'm going through my enzyme box and testing to find out which step got messed up.  This should only take a couple of days, but re-doing those timepoints is going to be a complete pain in the ass.  It took two weeks the first time.  I'm wondering if maybe these samples can still be salvaged though.  If one of the enzymatic steps in library creation failed, then the DNA in there should still be viable, all I need to do is redo that step with new enzyme.  I'm probably going to end up redoing the timepoints anyway for replication purposes, but its a total bummer that I've probably lost a month's worth of work to this.