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Suzy CA USA

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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Awesome Stuff
Tuesday, February 1, 2011

My grad schools days are long behind me, and as time goes on, my memories of the pain and suffering become more of a blur. But, there was one day that I will never forget.  Not only did I lose an entire days work, but I was lucky to avoid serious injury. It was the day I made the biggest mistake I ever made in the lab.

My graduate school was in a southern area of the country where obnoxious humidity hovered over you, causing you to sweat just from the act of breathing.  Homes in this state were built during the confederacy, and as such, did not have air conditioning. In fact, running an air conditioner, if you actually could afford a window unit, would use so much power that you could turn nothing else on in the place without blowing two of the four fuses which powered the apartment from an outside fuse box.

So, just like all good graduate students, I spent most of my weekends in the lab where the air conditioning liberally blasted away with no concern for the electric bill. Because of the unbearable humidity, and because I rode a bicycle four miles to the lab in this sweltering heat, typical clothing was always shorts, t-shirts, and plain white keds with no socks.  I was dripping sweat on arrival but achieved immediate relief once inside the arctic air of the medical building.

On this fine day I was alone in the lab, which I always loved and still do to this day. I was sequencing a 100 kb piece of chromosome six which we had cloned and identified as a segment of DNA commonly deleted in acute lymphoblastic leukemia (and some melanomas).  The hypothesis was that a tumor suppressor gene for leukemia was lost when the deletion occurred, causing cells to proliferate abnormally. Leukemia, essentially, is the proliferation of one of the cells that makes up the blood system, but it is not mature. The cell becomes stuck in an immature and nonfunctional state. It replicates out of control and fills the blood with white cells that don’t work.  Cures for leukemia focus on finding ways to push the cell out of its immature state to a mature state, or killing cells that are growing faster than the normal cells (this is why people lose their hair on chemo, because these are fast growing cells), or, just obliterate the entire blood system all together and get a brand new blood system, aka bone marrow transplant.

No one had ever found a tumor suppressor gene involved in leukemia. We thought it was going to be found in this region of DNA and that maybe, if we sequence this entire 100 kb piece of DNA, maybe we would find it. Crazy project right?? No kidding.

I did all my own sequencing by hand, acting like a human thermal cycler, rotating tubes between different blocks, mixing them carefully with the addition of radioactive S35 dATP and dideoxy nucleotides, and then stopping them precisely. I was pretty awesome at it. I could get read lengths of 600-700 bp using hand poured slab gels.  The data was translated by reading the bands on film on a light box and written on a piece of paper. All of that sequence was then entered manually into BLAST for alignment.

As was typical, I ran two large gels side by side.  The whole process from start to getting it onto a piece of film for incubation for 7 days took an entire day.  Here is what they looked like.

sequencing gelsequencing film

These gels stood vertically and sat in a buffer chamber on the bottom and the wells were sitting in a buffer chamber at top.  Once the gels are finished running, you would drain the buffers (which are radioactive) and then take your gels off the rig, separate the thick glass panes carefully so as to not tear your gel, and then expose it to film.

Well, I had been doing this technique every day for months, two gels a day (8 samples a gel) and was trying to crank through this data (because the cure to cancer was depending on it!).  Because I could do this technique in my sleep, this day, I didn’t pay attention to something very important.

Now, I was wearing a lab coat, because this is radioactive work, but just my little keds and no socks. The gels had finished and I removed the clamps that held one of the gels to the rig. We used grease to seal any leaks between the top and bottom, so the plates were still sticking to rig. I didn’t even think. I turned my back to it for just a moment. Just one moment.

The next thing I heard was the sound of smashing glass. I felt glass spray my legs, my feet, and glass shatter on the floor. Radioactive glass was everywhere. Including my shoes and in every crevice of the lab. The heavy glass plate sandwich unstuck from the rig and toppled right over onto the floor, behind my back.

When I think about that day, I just can’t believe how lucky I was that the plates didn’t slice open the back of my legs. When I looked down I could see large sharp slices of glass on the floor, much too close to my legs for comfort. People came out of their labs to see what the crash was. They felt bad but since the floor was now radioactive, no one dared come in.

I called home to ask my guy to bring me a new pair of shoes and socks. He did so begrudgingly, as I recall. He was probably high as a kite (his norm on weekends) and not into the bike ride down to the lab.

I cleaned up all the glass and then used the Geiger counter to inspect for radioactivity. It had to take me at least an hour or more to clean up the radioactivity from the floor. Then, do swipes and count them and re-clean, until there was no detectable radioactivity anywhere.

And for the glass in my shoes, well, I threw those away, washed with the decontamination cleaner, and didn’t worry about it. No point in worrying about something that you can’t do anything about.

Honestly, I was most bummed about losing all of the data.

I never, ever, took the clamps off the gels again until I was ready to move them and never turned my back on the gels again. It is very easy to get so comfortable in the lab that you forget what you are doing is actually dangerous.  The mistake changed the way I did all my work. I had new respect for the dangers in the lab, and for the risks we put ourselves through to do our research.

Oh- and I always wore socks.

But I never did find that tumor suppressor gene…….it’s still waiting to be discovered. I just know it.

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Blog Comments

MitoScientist
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Arg, I feel your pain on the sequencing gels. I am in the midst of trying to get them to work with Alexa-fluores instead of radiation, to look at exact lengths of poly(A) tails. They are such a bitch to set up, our casting system is from the 80's I swear, and it drives me absolutely nuts.

Also, I would think the situation of a radioactive spill would hopefully make any significant other not whine so much when help and or shoe retrieval is required!


Suzy
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Hi Mito- wow- really? You're using that old equipment? I am so sorry. Yeah those things are a mess with unpolymerized acrylamide dripping everywhere until it finally sets.

The event was so traumatic, I can't clearly remember that part of how I got new shoes and talking to my guy. Just that there was a hassle about it on the phone.

One thing I DO NOT MISS about working in academics is the amount of radioactivity I worked with.


MitoScientist
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Well to be honest, I'm sure the casting equipment is much newer than the 80's, but its definitely not from the 21st century. I think my fluorescence will work in place of the radiation, but the casting and running of the gels is definitely a beast to be tamed. And our lab isn't big on new equipment spending, so no hope of buying a new one from Bio-Rad or something. So its up to me to cobble together solutions to leaks and whatnot! I hope I look back on all this and laugh, from a well-funded lab in the future!


Brian Krueger, PhD
Columbia University Medical Center
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I hate those gels, Jade.  I was using them to do footprinting assays in my PhD lab.  They're such a pain in the ass.


David Manly
Freelance Science Journalist
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Oh gels ... how I despised them!

And then, when you get them to work for the very first time, what an amazing feeling! Sadly, that feeling disappears on your 1,000th gel :(


Geeka
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I had the same problem a few times. I had it so many times, that my boss decided it was cheaper to send the stuff for sequencing rather than buy new glass plates. I think that it's the gaskets that ended up being bad.

I was just on the cusp of when tech was really integrated into labs. Maybe we should do a monthly post on the coolest piece of tech/coolest thing you could outsource when you were in grad school?

 

On a similar note, the woman who got booted from the lab before I started exploded a 15ml conical w/ p32 in it all over the lab. I looked up one day, and rad safety had exacto-knife'd pieces out of the dropped ceiling.

Tideliar

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Wow, excellent post Jade!


Suzy
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@Mito: Try vaseline to seal the gaps and leaks. I think that's what we used and it works well. We used the large black binder clips to seal the gel down the sides on the gaskets while the acrylamide was polymerizing. They are super tight. I think the fluorescence will work but will you capture the signal by hybridizing after running? I wonder how much light exposure the fluorescent method can take before the signal is lost.  The radioactive method still took 7 days to develop so anything should be better than that.

@Geeka: a coolest piece of tech post could be fun. I'll start thinking about it... I've never had an accident with P32. We had a very healthy fear of it. There was no ever being complacent with that stuff.

@Tideliar: Many thanks, kind sir!


Gerty-Z
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Nice post. I HATED the manual sequencing. I remember when we could send out sequencing. it was an awesome day. I will always have anxiety dreams about bubbles that would invade those gels, the really old, rusty binder clips etc. Good times :)


Suzy
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ha ha- exactly! The sequencing core was pretty new so we didn't use them when they started up. They synthesized primers (before they did sequencing) and people always complained about the quality, so I think it was assumed the sequencing quality would be the same.

I wish I had investigated it more. I bet the sequencing kits and radioactivity cost more than the core.

I wonder what methods students do today will become obsolete and replaced by something far superior in the future? Next gen sequencing is already replacing Sanger.

 


MitoScientist
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@Jade: Yeah, I've gotten them to work, the vasaline had dried out and the caster definitely worked better with a fresh coat. As far as the fluorescence, we're actually using Alexa-labeled primers for our specific mRNAs in order to detect the tail, and so far it looks like it will work well enough to replace the radiation normally used in the assay. With the current alexa-fluors, they don't lose too much signal from light exposure, at least as they're used in our protocol. Pretty happy to drop 32P from the things I have to keep track of and handle on a regular basis!

Chem Jobber

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There's nothing like molecular biology techniques to make an organic chemist feel like their work is a cinch.

BERNARD DELACRUZ

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Bad: screwing up your sequencing gels. Frown

 

Worse: knocking over your fellow grad student's sequencing gel. Yell


Suzy
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@Bernard That would be tough, but, shit happens. I hope they weren't too hard on you!

@Chem Organic chemistry can be dangerous too, no?

@Mito I am SO HAPPY not to work with P32 anymore. In my postdoc lab, we used radioactive iodine instead- a much stronger emitter. The signals come up much faster but you had to wear lead shielding to work with it. (But then waste was discarded down the sink so WTF?) I hated it.

 

Amie Moody

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Hi,

I'm a 5th year grad student and have been working with sequencing gels since my rotation.  Until very recently, I could pour and run them in my sleep.  Then one day, when I was pre-running the gel, about 30 minutes into it I saw that my wells had swelled out the top of the plates! I suppose in compensation for this, there was also a larger frown at the bottom of the gel where it was pulling up from the bottom.  Since then, every since gel I pour does this and I have not been able to track down why this is happening.  Furthermore, I can not think of and do not notice that I am doing a darn thing differently than I was when the gels behaved themselves.  Any suggestions would be greatly appreciated!

Thanks,

Amie


Suzy
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Hmm...ok.

So you are saying that the well itself is swelled- like the acrylamide is warping? What happens when you try to load the sample into the well?

Did you recently change batches of polyacrylamide? Or a component of your gel mix?

Has someone changed the settings on your powersupply?

Are the buffer chambers maintaining their volume?

Perhaps the plates are not being held tight enough along the comb when it is solidifying?

 

Let's start with these questions.

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