Chronological Lab Notebook

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