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Historic day

I have poked my last crickets. In Nebraska, at least.

Which means I don't have to get to the lab by 7 tomorrow morning, hurrah / phew! I am glad that part's over, and I will not look back.

Of course, today is the first day in around 6 months that everything in the lab is working again: running water (hot and cold, such luxury!), gas, compressed air, and the walk-in cooler.

I feel like this is not a coincidence.

However, it isn't as though the construction has ended. Instead, it has entered a new, diabolical phase where they are deploying some sort of massive grinding implement that shakes everything.

This makes it difficult to weigh things on the balance.

It is also the opposite of calming and is making it even more difficult than usual to concentrate.

Now, what's left. I need to catch up on data entry so I can determine whether or not I need to do anything further with the cricket ovary samples. I need to extract lipids from around 200 cricket ovary samples, 24 samples at a time. I also need to do an oodle-ton of data analysis on this oodle-ton of data.



( 9 remarks — Remark )
Nov. 5th, 2015 04:43 am (UTC)
So how do you do the lipid extraction? Sonicate them? Waring blender?
Nov. 5th, 2015 02:12 pm (UTC)
For the ovaries, I sonicate the ovaries in a chloroform-methanol solution. There are some interesting logistics involved. Sonication causes the chloroform-methanol to splash around, so I wound up modifying a set of eppendorf tubes by punching holes in the lids with a leather punch. I use those lids on the tubes that contain the ovarioles, which reduces but doesn't eliminate the splashing.

Another tricky/tedious part is removing the solution from the remaining ovariole material. If the first lipid extraction isn't complete, the ovaries wind up floating on top of the solution after sonication/centrifugation, so I have to use a pulled pipette and work it under the ovariole tissue to slurp up the solution and put it in a separate tube. If the tissue is really chunky, it clogs up the pipette and the transfer takes a while. Then I repeat this process with a second chloroform-methanol wash, and at that point the ovaries typically sink to the bottom of the tube.
Nov. 6th, 2015 02:42 am (UTC)
Man, what a pain, and what a lot of tedious, delicate work!
Nov. 6th, 2015 03:03 am (UTC)
I think that might explain why I put off working on that particular task today.

At least I don't have to solubilize the remaining tissue after that, which adds two more days of work to the sample-processing! (needed to do that for glycine-injected crickets to quantify how much glycine is used to build protein)
Nov. 6th, 2015 04:00 am (UTC)
What do you use to solubilize proteins without digesting them?
Nov. 6th, 2015 05:37 pm (UTC)
Well, the process basically digests them - some sodium hydroxide plus some proprietary blend of chemicals from Perkin-Elmer. The more important part is that before solubilization, I rinse everything with two swishes (scientific term) of 10% trichloroacetic acid, which precipitates out protein but leaves any unincorporated glycine in solution. Then I know that any radiolabel that's detected in the remaining (solubilized) solids has been incorporated into protein.

Oh, and then after solubilizing the proteins, they get decolored with some fizzy 30% hydrogen peroxide. Colored compounds interfere with detection of the radioactivity ("quench" it).
Nov. 7th, 2015 02:29 am (UTC)
Oh, cool, that makes a lot of sense. The TCA clots the proteins. And yeah, I've played a bunch with lasing media so I'm familiar with quenching. I'd never considered that you could just bleach it before.
Nov. 8th, 2015 02:56 am (UTC)
I'd love to take credit for the cleverness involved, but I'm no biochemist. When working through the initial phases of this stuff, my boss said that TCA is often used for wart removal, interestingly.
Nov. 8th, 2015 05:38 am (UTC)
It is, although I'm not sure there's a ton of research backing it up over, say, HCl or cryo. I'd read about it originally in that context, as a way of changing pH by changing electronegativity.
( 9 remarks — Remark )

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