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Brian Krueger, PhD
Columbia University Medical Center
New York NY USA

Brian Krueger is the owner, creator and coder of LabSpaces by night and Next Generation Sequencer by day. He is currently the Director of Genomic Analysis and Technical Operations for the Institute for Genomic Medicine at Columbia University Medical Center. In his blog you will find articles about technology, molecular biology, and editorial comments on the current state of science on the internet.

My posts are presented as opinion and commentary and do not represent the views of LabSpaces Productions, LLC, my employer, or my educational institution.

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Wednesday, July 13, 2011

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.

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JaniceF
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I feel your pain.


Brian Krueger, PhD
Columbia University Medical Center
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I redid the adapter ligation and PCR today on some of the samples and the smears are super bright now.  So either it really was the adapter ligation that failed OR running another 18 cycles of PCR just amplified what little product was there.  I didn't do a "no ligation" control, so I'll run PCR on a sample I didn't religate and see what happens.  If the smear is still crappy looking then I know the adapter ligation failed and my redo is good :) But I'm kind of pessimistic, so I'm betting my new good looking smear is just because of PCR, and I'm going to have to redo everything.


Dr. Girlfriend
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Stick more passive-aggressive “please make sure the freezer is SHUT” notes on the door! Every lab I’ve been in has this problem (and multiple capitalized notes on the door). Such a simple thing causes so much damage – lost experiments, damaged enzymes, and pissy lab mates - There has to be a solution.


Brian Krueger, PhD
Columbia University Medical Center
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You mean like the one that's been on there since the first day I got the freezer?


Alchemystress
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This totally blows.. sorry to read this

 


Brian Krueger, PhD
Columbia University Medical Center
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My "no-religation" control amplified too.  So now I don't know what got messed up...

FunkDoctorX

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Wow, that totally blows.

 

As for signs on freezer doors...I think they are only effective for about a week or so after they are put up. After that, it just blends into the background. If you wanted it to be truly effective I think you would need to have the sign changed every couple of weeks, put in a different spot, use a different font, say something funy etc...but who the hell wants to do that as a lab job (although it's better than other lab jobs for sure!).

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