Syntax Is Important

Carebear (our latest PhD student) texted me this morning to let me know the experiment was on, and tell me that the “laminin is in the bottom of the -80 on the right side.”

We have two -80 freezers. I opened the one on the right, a little bemused since it's full and we don't go in and out of it much, only to discover that it was obvious no one had opened this freezer recently… It took two of us technicians five minutes to reclose it.

The laminin was on the bottom right side of the left hand freezer.

7 Quick Takes

1.  I hate everything about my job right now.  The stress levels.  My son sobbing when I leave in the morning (even though he gets to stay with Daddy every day).  And especially that my *redacted* genotyping PCR refuses to cooperate.  Someone please explain to me why a PCR protocol that has worked seamlessly for me for years (and still works seamlessly for other people in the lab) can just stop. working.

2.  Everything went pear shaped yesterday around eleven in the morning, and, on top of all the stuff scheduled for Thursday and the stuff I was making up from Wednesday, when I was randomly sick with a GI problem, we decided to do an impromptu 3 mouse adult cardiomyoctye isolation.  So I was stuck there till almost 6.  Then on the way home, my bus got out of downtown and onto the HOV lane, and just stopped – way up ahead another bus had stalled and no one could get around it.  They had to back a wrecker for miles down the HOV lane to get it out.  Thank heaven I used the ladies room before leaving work.

3.  Remember the rotting trim issue?  That it wasn’t the doorframe, just the trim.  When we took the trim off, we found the house frame itself had started rotting.  There wasn’t very much damage at all, thankfully, but you could see it was starting.  Turns out my disgusting experience of jamming my thumb straight through rotted door trim was a luck break, we caught it early enough for Himself to fix it.

I’m incandescently angry at the builder though.  We’ve had no flooding.  The house isn’t even three years old.  There’s NO REASON for this.  Well, there is a reason for this, mostly the builder thought it was a great idea to create an area with a door coming out of a bay window that creates an area where water pools by the house and can’t drain.  We’ve got to fix that next.  Then the next major project, before replacing the downstairs carpet with flooring, adding cabinets to the kitchen, or even putting up my honeysuckle trellises, has got to be putting a properly graded patio and pergola off the back of the house.  To keep the water away from the house.

4.

Is this not the cutest photo ever?  (Yes, we let the not quite four year old hold the baby.  He loves babies.)

In fact, all the discussion of babies, brought to the forefront of his little mind a promise I made months ago to make him a toy pouch sling for his baby doll (which is naked, because I threw out all the pink stuff it came wearing).  So now he’s nagging me.  But he’ll have to wait until I’ve finished the roman shades for the breakfast room.

5.  No, the roman shades aren’t done yet.  I’m having technical difficulties with the batik.  Plus I’ve been busy.  And I’m pregnant.

6.  I also haven’t gotten my chandelier spraypainted yet, although that hold up isn’t my fault.

7.  …I’m cleaning my pantry and I just found a big stash of bakers’s chocolate on the top shelf.  And it expires really soon.  So, in the interests of frugality, I have to go bake brownies now.

…AAH  We don’t have any EGGS.

For more Quick Takes, visit Jen @ Conversion Diary.  Maybe she has eggs.

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?