Bad days aren’t always our fault.

I have been having PCR trouble for two weeks now. It’s driving me crazy. One day, the assay worked, the next it didn’t. I’ve been repeating them for weeks, sure it was the dNTPs, since we weren’t using our usually brand, and that was the only reagent I’d changed.

The good news is that it wasn’t my technique. The bad news is that the machine is broken.

The way quantitative PCR works is you add a fluorescent probe to the normal PCR reaction. The probe binds to the middle of your DNA strand and is broken into bits when the taq enzyme replicates the DNA strand. Breaking up the probe separates the fluorescent reporter molecule from the quenching molecule, and the assay emits fluorescence detected by the special (read expensive) PCR machine.

Well, this doesn’t work when the lamp is broken. Not burned out, broken. So we have to get it fixed.

But this has been driving me to distraction for a fortnight, and affecting everything else I did. I’ve messed up genotyping, I’ve locked my keys in the truck at the gas station, I burnt the crust for my lime tart. It’s all been bad, just because I was obsessed with figuring out what I was doing wrong. And equally obsessed with why I could never seem to do anything right.

When Noodles (our postdoc) told me he thought the light was broken, I ran upstairs to ask the only other recent user if her plates had been working. And the tone of voice when she said no and the expression of relief when I told her that we thought the machine was broken told me she felt the same way I did.

It’s a hard thing to remember that sometimes you aren’t doing anything wrong when an experiment doesn’t work. The immediate instinct is that you have screwed up. When you can’t figure out how you are screwing up it’s extremely distressing. And when you realize you aren’t the problem it’s such an incredible relief it’s like falling up.

Just Some Ranting

Well, if I’m lucky (in this specific context lucky means the PCR machine gremlin will not manifest himself) I can leave to go home at 7. Yes, that’s 7pm.

Everything has gone wrong today.

My RNA samples wouldn’t thaw, and I had to dilute them before I could pipet my plates.

My first plate’s RT mix was short by two samples, and I had to make up another batch, during which I ran out of a reagent and had to thaw a fresh tube.

Then the PCR machine’s screen for the RT step borked and I had to start it over.

Then I volunteered to do a transfection tomorrow so that our PhD student could spend the day with her parents and not worry. I have to come down here anyway to get my macula checked, so why not, it’ll only add another hour.

But that got me roped into doing the transfection today too.

Only first I had to move my first RT plate into the qPCR machine, so I start making my PCR mix, only to find I need to dilute more forward primer and probe.

Then I can’t find said primer and probe.

When I find the probe, I can’t get the blasted box open, it’s frozen shut.

Then they have to thaw.

My PCR mix is short by one sample and I need to make more. During which I run out of MgCl2 and need to thaw more.

I drop the cap of the brand new MgCl2 on the floor, so I need to decant the whole vial into a clean tube.

All of this getting my plate ready delays starting the transfection, so instead of 3:30, it started at 4:30.

I come downstairs and get my second RT ready for the PCR step, and this goes smoothly. The way everything else has gone today I’m sure I did something wrong, but I’m not sure I care. This plate won’t come off the PCR machine till 7 pm, and I have to stay to turn the machine off.

So I’m chilling at my computer and blogging. I should be measuring more histology, but I’m tired.

And with the boys in the valley visiting Mike’s parents, it’s not like there’s anything waiting for me at home anyway.

Well, there’s leftover cheese pizza.

Incomprehensible

Thursday was a bewilderingly annoying day at work, courtesy of our MD/PhD student and our 4th year medical student on rotation.

Between the two of them, between their two undergraduate degrees and 7 cumulative years of medical school, they could not make and pH a buffer with instructions, in under 3 attempts.

First they came and asked me where the 1M TrisHCl was, I check for them and told them they needed to make more.
Then they needed instructions to make more, which I provided. Making a 1M solution should be trivial, but not everyone paid attention in Chem 101, so hey. You weight out the necessary salt, dissolve it in less than your total volume, pH the solution, then bring it up to the total volume.
Then they needed help working the pH meter – fair enough, our pH meter is pretty obnoxious. I go and show them exactly how to calibrate it and then we try to pH the buffer they made.

• Problem 1 – They only made 10 mL of buffer. Seriously, why wouldn’t you just make 100 or 200 mL of buffer? It won’t go to waste. This is more a peeve against selfish laziness.
• Problem 2 – They didn’t dissolve the salt. I kid you not, they handed me a 15mL conical tube with a lump of undissolved salt in the bottom.
• Problem 3 – Prior to pHing, the buffer was already over the maximum volume. They had 11 mL of buffer, it looks like they put 10 mL of water in the tube and added the salt to that. This makes the solution 0.91M instead of the 1M they needed. I specifically told them how to avoid this when I gave them instructions, and they ignored me.

At this point, I step in, weigh the necessary salt for 100 mL of buffer, put it in a bottle with about 75 mL of water and a stir bar and tell them to pH it with this 1M sodium hydroxide [at this point I displayed the bottle] when the salt is dissolved, then don’t do anything else until I get back, because I have a delivery to pick up at the loading dock.

When I get back, they’re running water into the bottle… It turns out they tried to pH their buffer with the calibration buffers for the pH meter.

The pH calibration buffers are brightly colored.

I presume they got it right the third time around, but I had an experiment to start, so I don’t actually know they did it right. I do know that I will not use any of that buffer, I will make my own.

I’m not annoyed that I had to tell them how to make the buffer. I’m annoyed that I gave them step by step instructions and they still couldn’t do it right. And it isn’t an isolated incident. We go through the same thing with how to do Western blots, extract protein from tissue, and on and on and on, over and over again.
If you cannot handle some of the most basic laboratory techniques after two summer rotations in our lab, followed by being shown by the senior PhD student and the postdoc, not to mention myself, what are you doing getting a PhD?
The 4th year medical student I can at least understand, a month long rotation doing basic science research is a cake walk compared to clinical rotations, it’s almost a vacation. You can’t get anything done in a month, so there are no expectations. It’s just C.V. padding.
But if you can’t do the benchwork, even with multiple explanations of how the technique works and what to do, why are you torturing yourself?
Better question, why are you torturing the rest of us?

Problem Solving

I love elegant solutions.

This past week, I threw myself into solving the problems I’ll encounter going back to work, and I’m really satisfied with my pumping solution.

We’re moving, and instead of a 30 minute walk/bus ride, I’ll have an hour bus ride to and from work. I must schlep briefcase, purse, lunch, breastmilk cooler, and pump, which is an awful amount of stuff to schlep.  Oh, and I’m on a budget, I need to do this as cheaply as possible.

So, for pumping at work, I needed a pump, something to pump into, and transportation solutions for both. I bought the Lansinoh double electric because it was the cheapest good pump. Bonus, it didn’t come with a huge ugly tote to schlep. Malus, I needed something to carry all the fiddly bits in.

On impulse, I picked up this train case from the Container Store.  I wasn’t sure the pump body even would fit, but everything fits as if it was made for it!  The tubing goes in the zippered mesh compartment of the lid, where it can’t be damaged, the pump base and adapter go in the bottom of the main compartment with the flanges and two collection bottles on top.  The diaphragms and valves go in the zippered flap pocket and sit on top of the pump and flanges so they won’t be damaged.  There’s a gusseted inner pocket where I can keep a soft towel for drying off the parts.  I can even keep a Sharpie for dating storage bottles in one of the brush pockets.

I picked up a small cooler for ten bucks from Babies R Us.  My hands-free solution was free.  I already had storage bottles.  My total cost was approximately $180, fifty smackers less than the cheapest equivalent setup, and with a smaller footprint.  I’ve done a few test runs and am extremely smug about it.

But I find that all the elegant solutions in the world don’t make me feel a whit better about leaving my baby.  I wish I could take him with me.  But even if such practice was acceptable and permitted, I couldn’t.  I spend too much time in the lab.

Non-native speakers don’t get tonal subtleties…

…including “I’m not going to discuss this.”

A full half of my lab grew up speaking some language other than English, and none of them (well, of those who are fluent) have trouble with this.  Frenchie or Noodles will occassionally ask me to explain some idiom or piece of slang, but they are perfectly polite and know better than to pry on rude topics.

Like “when are you going to have babies?”  Nosy just doesn’t get it.  If we get anywhere near the topic of children (unavoidable as about 50% of my coworkers have children) she asks me when I’m going to join them.  I don’t look at her, I change the subject, I even tell her point blank that I don’t want to talk about it (all Nosy does is ask why not) and I can’t get through to her.

And of course I can’t tell her to do something anatomically improbably to herself, no matter how emotionally satisfying it would be, because then I’m the one being rude and abrasive.

Frenchie, who knows I’ve had two miscarriages, tries to be comforting by telling me I’m still young.  And I know she’s trying to help on two levels, both heading Nosy off from her questions and reassuring me that I still have time.  But it just doesn’t help.  My very youth is part of what upsets me.

I am young.  And I had two miscarriages before I was 27.  How is that supposed to make me feel?  Intellectually, I grasp the statistics of it.  I know I’m at no greater risk of a third due to the previous two.  I’m thankful for my OB for giving me a gentle, but firm verbal smack upside the head whenever I get overly fatalistic in his presence.  Which I’m prone too, because I’ve had too many bad experiences sitting on a table partially or wholly clad in paper just in the past 14 months.  But I’m still young.  And in my view, that makes this worse, not better.  I should be at my healthiest, and something has gone wrong twice anyway.  How is getting older supposed to fix that?

I’m uncertain about many things in my life.  Where I’m going, how I’m going to get there, whether I even like where I’m going or how I’m getting there.  But I was never uncertain for a moment about having children.  Not that I was deluded enough to think it would be fun and easy, or a cheap way out of deciding where I’m going with my career.  It’s important to me because of who I am and it’s not something I can change.

And for the last year, I’ve faced the possibility that I can’t have what I want so very badly.  Of all the hard things in life, I never once considered this, why should I when my own mother had three of us after 30?  And it’s more miserable than I can communicate with words.  Because I have the sensation that I can’t ever have what I want, that I’m defective.  Because if there is something wrong, the doctors won’t look (and I won’t permit them to look) until after a third miscarriage.

Which means I get to sit here in uncertainty and be tormented by both rude and kind coworkers who respectively persist in wanting to know these intimate details or try to make me feel better by telling me I’m still young when it just makes me feel worse.