Chronological Lab Notebook

Cracking open some tobacco seed to TC tests I started in mid Jan. 3 diff media formulations.
Base media: 400ml water, 2g MS, 3.5g agar, 12g sugar. 25 ml / tub
Green: 25ug BAP, 3ug NAA (in 25ml)
Red: 6ug NAA
Clear: 25ug BAP, 6ug NAA
Seeds plated on clear, 3/tub transferred after a month or so.

Attempting tissue-culture-free agro transform of duckweed (lemna minor). I ran a few diff exps this weekend, and more excitingly noticed a red frond in some that I'd tried to tfm on Feb 9th, hoping that means the meristem took in the genes and this is a properly transgenic new frond. Rough protocol:
- A loopful of agro into infiltration mix, in a syringe with some duckweed fronds
- Pull back plunger while blocking end, and release. The sudden vacuum+slam (in my head at least) helps push agro into the interstices of the fronds.
Will share more depending on how more recent tests go.

• a couple of colonies visible by this evening on the electroporation plate(s?). Will evaluate tomorrow.
• went down the rabbit hole of DVD OPU microscopy options, AFMs, flexures etc again
• modelled and am printing bits for a nicer spectrometer

New run:
- Pseudomonas in liquid culture for ~18h
- Spun down, removed supernatant, add 10% gly (in 1.5ml tube)
- Spun down, removed supernatant, add 10% gly
- Spun down, removed supernatant, add 30uL of 10% gly (pellet by this stage was pretty small given my weak 'fuge)
- Added 3uL DNA (from a mini-prep I did yday, aeBLUE on a kanamycin resistant backbone)
- Did this in two tubes. One got 1.2mm gap, one got 2.4mm. 10 zaps each. On the 2.4mm gap, peak was ~5V (~10kV/cm). On the 1.2mm gap I didn't get a clear voltage reading, not sure what went wrong.
- 1h in 200uL LB broth @30C to recover before plating 100uL on Kan plates. Now we wait. Only worry: cells might have been stationary phase, might try a repeat with fresh culture in a few hours.

I ran another mini-prep, although since my centrifuge is funky I used a syringe filter after the precipitation step. Then to see if there is DNA there, I added 2uL SeeGreen stain to 20ml water. I add 200uL of this plus 2uL of my samples: known DNA plasmid at 10ng/uL, miniprep from ag, miniprep from ecoli last time, water (W) and the latest product, this time from liquid culture, e.c. with pBLUE. Did this first in micro centrifuge tubes and couldn't see anything past their own blue scatter/fluorescence. Transferred to glass vials and bingo, finally, a difference between the water and the others. I need to take some better pics, but it looks like they all contain DNA after all! And the color makes me think my fears when I ran the gel and saw the orange clouds were founded: the DNA prob just washed out of the wells. TIL add buffer first! As I was pouring the buffer I noticed surface tension or whatever meant well 3 was the last to fill, at which point I stopped pouring, prob why it was the only one that showed bands. Other likely contributing factor is my wimpy centrifuge not getting rid of as much residual ethanol from the DNA wash buffer during the miniprep, which can make it floaty. Anyway, at least I think I have DNA aplenty now to test electroporation and such with! Maybe in the future I'll build something nanodrop-like to quantify conc. or find a lab with one.

I was killing + throwing out a primrose used for testing, and there was one nice flower left, so I stuck the stem (split) into some water with highlighter fluid added. After some time, glow under UV spreads to the petals.

Adding vitamin C or citric acid gives a white (almost pink-white) glow, adding sodium hydroxide gives green.

I did a mini-prep on some ecoli, and had the result of a previous mini prep. I wanted to see how much DNA I had in each, so I ran a gel with these plus some reference plasmid DNA @ 10ng/ul. Sadly the only bands I saw were for the reference plasmid, so I'm doing something terribly wrong. Cool to see the supercoiled/nicked/linear forms of the plasmid spread apart at least.

I made some liquid cultures yesterday, and today am trying a wash step before electroporation. First test in the am:
- 1.5ml pp culture, spin down, remove supernatant
- Add 1.5ml water, spin down, remove ~almost all of it
- Resuspend pellet, ~30uL liquid total, and place on 1.2mm gap
- Add 5uL DNA (@10ng/ul)
- Zap 5-10 times
- Into 100uL LB broth, warm (~30C) for an hour, plate.
I like P. putida - it got cloudy much faster than e.c. (first pic shows after ~12h maybe?) and doesn't smell like poop. Will run more tests later today / tomorrow as time permits, I might also want to switch to DNA that has a visile marker, I ended up with single colonies on a few plates last time and I'm not sure if they are just contam or survivors that evolved resistance or if they actually took in the plasmid, maybe I'll mini-prep one of sebs chromoprotein colors so I have plenty of e.g. GFP plasmid for testing this. Nice Saturday project option.

Later: Hooked up my oscilloscope with a 10k resistor in series with a 5M to cut down the voltage, then ran it with a few configurations:
Open: -11V spike
Cells in liquid culture spun down once: sometimes a very short spike of a 1-2V
Cells in LC, spun down, resuspended in water, spun down, then in 30uL water: -5V for 5us (then a smaller +ve peak too)
Cells in LC, spun down, resuspended in water, spun down, ~all the supernatant removed, then suspended in 30uL 10% glycerol: -10V spike for 5us (then a smaller +ve peak too)
All this with a 1.2mm gap. Pinch of salt: get a spike of a few V even with leads disconnected, and 480k is a big load for a piezo. Still - clearly the washing is actually needed, I should probably do several washes next time I try this properly.

Doesn't look like the electroporation worked. I did get one bit of contam tho which is nice... (Maybe one colony on one plate is legit? Or maybe they need more time? But I suspect I need to re-try with more rigorous cell prep :/

I got excited when I noticed that the flowers which I had injected with the Odin's agrobacterium Ruby system had some bright fluorescence under 360 nanometer UV light around the injection sites. There was a little bit of red ruby expression too, as I expected, but nowhere had I seen anything about a fluorescent reporter. This was exciting because I had used some DNA that I had extracted via miniprep from the agrobacterium in a first YOLO test of my Gene gun. I rushed to fish the test onion slices out of the dustbin and put them under the scope with a UV light illuminating them. Sure enough on the positive sample that I had shot, I could see some vague blue glow in a few cells similar to the pictures I saw online for the original O'Reilly Jean gun instructions. Very exciting! Except that I didn't know why they should be fluorescent genes in this plasma. I asked the Odin people and they confirmed that it shouldn't have any fluorescence- it's just Ruby. I am now tasting on a fresh flower, just the injection media or just water to see if the fluorescence is a response to damage by the primrose plant- my suspicion is that the onion was also some sort of autofluorescence and unrelated to the DNA that we shot into it. Update: yup, just the injection media was enough to cause some bright fluorescence in the petals - false alarm.

I'm testing out a bunch of different combinations of parameters for electroporation both in E coli and in the pseudomonas species that I have. Trying variants with a 1.2 mm Gap and a 2.4 mm Gap. Trying room temperature versus chilled trying one spark versus three and doing it all in a sort of taguchi array.

I got a boost converter than can go up to 60V. Poured a 1% agar gel in a clear plastic tub, with carbon rods as electrodes. Ran some food coloring and some highlighter fluid to test. 0.2% sodium bicarbonate buffer. 40V. One binder clip touched the buffer and gave some yucky reactions. But the dyes did separate. Proper agarose, TBE and seegreen safe DNA dye arrive in a few days.

Until I retroactively add these experiments manually

Isolating a glowy pseudomonas, trying to lyse cells, pouring a few plates

• First gram stain
• Had various things growing