Chronological Lab Notebook

I found this old product at SCRAPS PDX. Its a stack: PET carrier, some sort of release layer, shiny blue + aluminium layer and some heat activated adhesive. OG use was getting selectively stuck to laser printer toner to give your printed pages some bling. WIth a little heat it sticks to PLA quite nicely, although even surface contact seems important.

So at least some seem to be making stable daughters :)
GOsh I have so much stuff I should document and make notes of but I might have to declare bankruptcy on some of them, I'm behind and need to do more new experiments.

My poor duckweed stock; I cleaned em out and then forgot to add nutrients (I think)? Just noticed the state of them. Added some quickly, more than usual, will see what happens.

Hacked a UV camera to work on my mac!
https://github.com/johnowhitaker/uvcam
https://www.amazon.com/dp/B0H15QWXWB

Last transform was mid-april, most of these are from older ones. I collected some red fronds together to show off, here's the collection. They don't thrive, but I think there are some new small ones? Any left along end up surrounded by green though. Got some new tips for transform to try.

Lemna major vs minor (I guess major is now Spirodela polyrhiza but still)

Separating out some algae (also starting to think about ordering some chlorella and spirulina for edible algae experiments). Sorting old duckweed, about to sort new and big duckweed from whitaker ponds slough. Some water mold on one old dish. Some glow in eyGFPuv dwe but mostly from damage, TBD what grows.

After switching from tween 80 to span 80 (lol) I can finally do water in oil droplets, even with this crummy test droplet generator. (5% span 80 in mineral oil for this test). Todo wider channels for the oil since it is more viscous. See X for video, mesmerizing

Droplet gen working, Todo note on tween 80 vs span 80,

Indoor plant yielded a nice purple tomato, picked a little early, seeds preserved and packagred to share. Remaining indoor plants went out to join the flourishign outdoor ones now that some seeds are in the ba nk.
Some seeds on tissue, some left in water for two days then dried on coffee filter.

Mollala river, first try panning

Lots more crystal fun documented on johnoslab, including fringes caused by blowing over the cooling+evaporating slide to change the rate of crystal growth

I heated a pellet of polycaprolactone (soft thermoplastic) and stuck it to my 3D printer hotend with tweezers.
I then held a small industrial diamond (there were plenty of loose ones in a pack of rock carving bits I bough recently) beneeath it and pressed it up into the still-soft plastic.
Then I had codex whip up a UI to control the printer via the octoprint API.
I can now scribe tiny patterns into a glass microscope slide, to steer crystal growth. First tests cooling + evaporating now :)

Magnet snap attachment thingee for my pipette bot

I ran a mini-prep today to get more of my miraculin + eyGFPuv plasmid from some e. coli that has it. Mostly following the bioland miniprep 2 kit, BUT:
- I start with a couple loopfuls of bacteria right from the plate
- I found that I can use a syringe filter (0.22um) in place of the first centrifugation step (getting rid of the goop)
- For the wash step, I made an adapter: syringe -> luer-to-barb -> bit of tubing -> barb-to-luer. Into this I could press the bottom of the column, such that sucking the syringe pulled the wash through
- For the final elution step, I used my dremelfuge since I needed what came through, then with the remaining lysate I did another go on the column. I tried (unsuccessfully) sucking the final elution buffer through into a 1.5ml tube with a needle + syringe. Then after I gave up and centrifuged it, I discovered that with a bit of force the syringe luer outer diameter just fits into the top of the column, so I can push it through and collect in future (tested with water, looks like it will work and is great apart from a little foaming/splattering).

All that to say, I am now convinced that you can do a miniprep with this kit without needing a vacuum manifold or a centrifuge or anything - just a syringe, syringe filter (useful for lots of other things) and maybe some type of tube adapter for the sucking part (but also TBH I think blowing through makes that obsolete, just blow through all the column steps). Hooray!

Does this help anyone besides me? Not sure, I think most people doing minipreps have centrifuges haha. But I'm glad I don't need to set up the dremelfuge if the dremel has something else set up in it.

I did a quick vibes-based agro transform last week. On July 7 I:
- loopful of cells into a tube w/ 50 mM CaCl2
- 100uL per tube (one for +DNA, one control)
- 1.5ul DNA (~500ng) to +DNA tube
- Freeze (as cold as possible, I added CaCl2 to freezer ice + a little 96% ethanol, -10 or -15C, froze pretty fast)
- Thaw 5 mins at 37C
- Add 200uL LB and leave for 20mins to recover
- Plate and spread on plates with Rif. and gent. (maintenence) + kan. (selection for my plasmid)

By the 9th no colonies were visible but the plates had spend some time cold. By the 12th the +DNA plate had 5+ colonies and a big spot of mold. The no-DNA plate had some other contam. This was all a rush job before a camping trip, and checked after we got back - not ideal haha. I streaked 3 colonies onto a fresh R+G+K plate on the 12th and now have plenty growing. I tried an infiltration in tobacco leaves and a yolo duckweed one (just using the dregs in the syringe against some tiny duckweed leaves) - will see if we get transient expression in the leaves, at which point I'll bank my transformed agro and try some proper transformations.

Not a fan of how many steps this is if we want newbies to be able to replicate - lots that needs to go right! Gene gun (order plasmid, shoot, select) would be a lot more accessible. Time to order some algae.

(Inspiration/better protocol: https://pikaard.lab.indiana.edu/protocols/protocols/agrobacterium-growth-and-transformation.html)

The glowy poop on a slide that had had water added and then been dried showed phosphorescence again once sufficiently dry. I placed the rest of the white in 3ml water, then filtered it .22um. a drop on a slide gave birefringent crystals

After a call with a new friend, I sliced the surface layer off the two carrot targets (eyGFPuv and RUBY respectively) to see if more transformed cells might be hiding beneath the surface. Sure enough, some red ones in the RUBY target and some glowy ones in the eyGFPuv one, and not v.v.!

Maybe success? Not happy with it and older than I'd like, could be something on the surface, Todo more

From the public collection area near spectrum sunstone mine