Payback's... to be expected.

You may recall my agonizing a couple weeks ago over destroying some cells for an experiment.

This week, I was showing my labmate some crystals I had growing.  The tray slips an inch in his hand and water splashes to the cover slip, washing off the best candidate I had.  Se la vie.

At least he's guilted into showing me how to run high throughput trays tomorrow.

Back to basics. Water.

Today I planned to do a titration curve with my protein and check some ph points for future experiments.

I took a baseline with the NANOPure (tm) water just to see how the pH meter worked. Apparently our water is pH 5.8 +- 0.3 pH units, depending on the mood of the pH meter. But wait, turn off the stir bar and it shoots up 0.5 units. And if you use litmus paper, it's pH 4.7.

So I can't trust the paper or the meter or the water. No one knows the last time the machine was checked (years), but it should be every 6 months.

Here's hoping I don't have bad news for everyone tomorrow.

Update: a bit of reading reveals that our water is probably good. Dissolved CO2 hits exactly 5.8. The high resistance of pure water (no ions) makes pH meters freak out. I'm not sure yet what is up with the litmus paper. Need to get a direct resistance reading off the machine (should be above 18Mohms I believe). See if there are other tests to do. Or just a technician.

http://www.millipore.com/references/files/pmc_url/$file/highpuritywater.pdf

Destroying other people's projects

One of my lab's soon-to-be-alumni came back in town this weekend to do one more experiment.  I was prepping some cells for her in what should have been a simple procedure.  Of course, I'd never done it before (like most things) and had gotten a 10 minute explanation the last time she was here about what I'd have to do. The experiment itself is about 15 minutes of prep followed by 2.5 hours of sonication, then 10 minutes of post.

Of course, I messed it up. Everything was great except I forgot to turn on the chiller to the sonicator. Come back two hours later and the cells are cooked to a sterile 60C+.  There was condensation everywhere and things were nearly too hot to touch. Since we're not exactly engaged in protein folding, there wasn't much useful left of the cells. I flash froze them anyway just in case.  (Also to play with liquid nitrogen (sssshhhh)).

I tried to acquire new stocks from our partner lab, but the best they could do is start growing new stocks, which won't be ready till next week. So lab mate gets a vacation this weekend instead of working.  I don't think she was particularly disappointed by this turn of events, because now someone else gets to do her experiment for her.

This kind of thing actually happens all the time, but this was my first time in this lab.  Usually you screw something up yourself, at least until you have enough experience or have good "lab hands".  This particular incident was more unfortunate since she had flown in, but even having blown away $300 in tickets and cell preps, everyone pretty well blew it off.  Just have to try again next week.

Yay Mondays!

One strange facet of experimental science: if you time it right and leave an experiment running over the weekend, it's exciting to come in on Monday and hope for data!

Of course many experiments require monitoring (boo working all weekend). Fortunately, Crystal trays are intended to be left alone for...well, however long it takes. Years in some (extreme) cases.

I will have to find more projects that need to be left alone for a few days before they yield results.

Academic Code

Trying to edit some simple 2,000 line python code that was hacked into existence by 3 biologists who don't know an object from a hummingbird is the definition of academic hell. Fortunately, it's only 2k lines, I can fix it all in a couple days. Also, if you guys ever find this blog, I love you guys.  Just please let me teach you some basic programming best practices. :)

Friday thinking time?

I need to stop starting projects at 4 pm on Friday. Ones that take 3 hours to finish. Is so tempting to think, "oh but I'll have results by Monday!"

Should be Friday afternoon drinking time.

Cat Whiskers

I'd thought I'd remain far from the realm of animal research here in crystallography land. I don't oppose it, but don't have the constitution for some of it. Today I discovered that we need cat whiskers for a couple specific techniques. I was thinking for a while they were some kind of plastic filament, but no. Actual whiskers off a cat.  After being traumatized about this for a bit, I learned from my lab mate that the whiskers are reusable (unlike everything else in this field, but that's a different rant). Also he harvests them from his own cat, who tends to leave them on the bed while sleeping.

Preparing a dilution series of the seed stock makes it more quantitative, but by far the easiest and fastest method for seed transfer is streak seeding. This method uses an animal whisker (usually cat or rabbit) as a seeding wand, which is touched or stroked over the surface of the parent crystal to dislodge and trap the nuclei.  The whisker is then drawn through the new drop, depositing the seeds in a streak line. Although seeding wands have been made from thin glass rods, platinum-wire inoculation loops, and acupuncture needles, animal whiskers or hairs offer a definite advantage. The overlapping cuticles capture the seeds effectively. Here we would like to bring attention to an animal source of material for making seeding wands which, at least to our knowledge, has not been exploited much by the crystallization community.  Horse tail hair is much more abundant than most animal whiskers, which makes it useful when organizing large scale laboratory sessions.  In addition, horse tail hairs maintain the same thickness throughout their length (which is why violin bow makers use it), and this property may be useful in its own right for reproducibility in seed transfer. On the other hand, a cat whicker can be cut into three or four segments to obtain wands of different thickness.  We use both horse tail hairs and cat whiskers because of their complementary properties. We find that rabbit whiskers, being thinner than both horse hair and cat whiskers, are too flexible.

--Therese Bergfors. Seeds to crystals. Journal of Structural Biology, 2003.

Learning from your mistakes, directly

Checked my first crystallization plates today. Lots of precipitant; 70% methanol was not the answer (only 1/4 of wells down, no biggie). No crystals in most of the other wells yet, except one. One that I messed up.

That particular well I didn't seal very well, so it dehydrated. This concentrates the protein, inducing crystallization as the solution grow saturated. These might be usable for microseeding future trials.

The other wells may still form crystals, given some time. The other answer may be to poke holes in the seals and let them dehydrate too. The real answer is do it all over again and use a higher concentration.

Being science, will try all of them, as only one really needs to work.

The happiest place on earth

Today I head up to get my badge to the happiest place on earth, the synchrotron. Many shouts or eureka! And no small number of mumbled "that's funny"'s.

Milestone: Potato is Sequenced

In amusing science news, scientists have sequenced the potato!

• 884 megabases, or roughly 1/4 of the human genome, at 86% completion.
• 40k predicted genes, which is a bit higher than that originally predicted for humans, and twice what is currently accepted for humans. This number was confirmed by RNA-Seq, meaning these genes were actually expressed as mRNA, so are likely real.
• About 25% of genes are alternatively spiced, compared to 50% of human genes.
• two genome duplications
• autotetraploid, so have 4 copies of each chromosome, leading to some difficulties with breeding
• 3k genes which define the potato family 
• "acute inbreeding depression", meaning it's suffering intense problems like disease susceptibility because of how long we've been mono-farming the original few strains imported to Europe from South America.
• >62.2% of the genome is repetitive and will likely make the remaining 14% of the genome hard to sequence.

There are of course many other bewildering things about the plant, as seems to be the case in general in plant biology. Mendel was very lucky to study the particular traits in a particular species that managed to behave according to simple (Mendelian) genetics.  Many other plant quirks take some getting used to.

The experiments begin

Yesterday I started pipetting! First time in 4-5 years. So far I've managed to acquire most of the basic instruments, have a bench and fridge space, pulled a few chemicals out, and started mixing. Spent a lot of quality time with the micro pH machine getting my samples titrated. I spent even more time figuring out how to recalculate pH from several conditions, and eventually got it down well enough to write some code to do it for me. Now I need to go back to the pH meter and see how close I got. Monday I get to throw it all in crystal trays and the the real test will happen, though no results for a week or so. This is a slow feedback loop. Nothing else too exciting today, but hopefully good times this weekend.

Location:7th Ave,Oakland,United States

So many stupid questions

For the past couple days I've been struggling with noob questions. This is to be expected, but not questioning your whole understanding of acid/base chemistry. I found several protocols for crystalizing my protein, all fortunately starting with just ordering it from the Internet, as my protein is readily available. (usually there is weeks to months of ramp up time depending on what you can borrow from other scientists.)

The next steps are to dissolve the lyopholized protein powder in distilled water, then adjust the pH to 5.5 using NaOH, also known as sodium hydroxide or lye. A strong base... pH 14 at 1M concentration, for those who forget how pH works. Now, normal water starts at pH 7, so how am I going to add 14 to 7 and get 5.5?

I thought I had the answer as I'm also supposed to mix in 50% methanol. pH is only well defined in water, so I spent hours looking up how organic solvents affected "apparent" pH and doing a deep literature search and generally questioning my sophomore chemisty.

Finally, team leader reads the methods, laughs at me, and points out that proteins are quite acidic and I had to compensate that. I had briefly considered this, but naively assumed that they wouldn't be THAT acidic. Shows what I know.

Er, trust me, I'm a professional.

Better Living Through Chemistry

Welcome. I should begin in the grand tradition of first posts by laying out my grand vision of what you'll find in these pixels.  Really, it'd be as much to my benefit as to you. I'm curious to see what comes of it, or if it lasts more than a month.  I have a personal homework assignment of posting at least once a week, which definitely is to my benefit.

Last week I started a postdoc with [prestigious lab] at [even more prestigious university]. Astute readers will likely be able to sort out these details. I'll preserve my anonymity beyond that as I was raised in the internet age. This is going to be a professional blog and shouldn't contain anything incriminating or indiscrete.  But you never know when your coworkers will take offense at you pointing out their foibles to an audience of at least...ones of your friends.

Back to the prestigious university part, if you can't figure out who I am, you can at least trust that I am very good at what I do, even if I don't know much about what I'll be doing yet. I'm also very good at figuring things out. That's half the fun. So my opinions on the matters at blog will be at least reasonably well informed or annotated for speculation. At least in regards to science.

"I believe that a scientist looking at nonscientific problems is just as dumb as the next guy."  -  Richard P Feynman

Back to the vision, I'm going with three parts:

1. Talk about my daily experience with science, especially adapting the the new position and relearning my field.
2. Talk about science in general, and what's cool in the world.
3. Talk about the sociology of science: how it's done, how it's perceived, and whether it works.

There are innumerable blogs on these topics already, you are by no means obliged to care about this one.


Adios,
-Alex