The first thing we did was to filter the water samples with a membrane filter that is so fine that not even bacteria can pass through, but water can.
Therefore, after filtration, we would have all particles such as bacteria and larger dirt pieces in the filter.
From the 1 liter in each bottle, we only used 400 ml because previous experiments showed that filtering the remaining 600 ml is very time consuming because the membrane filter gets clogged up.
Therefore, the filtration step allows us to reduce those 400 ml volumes to more manageable Eppendorf tube volumes: Figure 1. "An Eppendorf tube". Reagents are expensive, and lab bench centrifuges are small!
Figure 1.
An Eppendorf tube
. Source. They are small, convenient and disposable.
Figure 2.
Labelled Eppendorf tubes on a rack
. Source.
Since the filter is so fine, filtering by gravity alone would take forever, and so we used a vacuum pump to speed thing up!
Figure 3.
Peeling the vacuum pump filter protection peel before usage
. Source.
Figure 4.
Placing the vacuum pump filter
. Source.
Video 1.
Pouring the water sample into the vacuum tube and turning on the vacuum pump
. Source.
With all this ready, we opened the Nanopore flow cell, which is the 500 dollar consumable piece that goes in the sequencer.
We then had to pipette the final golden Eppendorf into the flow cell. My anxiety levels were going through the roof: Figure 4. "Oxford nanopore MinION flow cell pipette loading.".
Figure 1.
Oxford nanopore MinION flow cell package.
Source.
Figure 2.
Oxford nanopore MinION flow cell front.
Source.
Figure 3.
Oxford nanopore MinION flow cell back.
Source.
Figure 4.
Oxford nanopore MinION flow cell pipette loading.
Source.
At this point bio people start telling lab horror stories of expensive solutions being spilled and people having to recover them from fridge walls, or of how people threw away golden Eppendorfs and had to pick them out of trash bins with hundreds of others looking exactly the same etc. (but also how some discoveries were made like this). This reminded Ciro of: youtu.be/89UNPdNtOoE?t=919 Alfred Maddock's plutonium spill horror story.
Luckily this time, it worked out!
We then just had to connect the MinION to the computer, and wait for 2 days.
During this time, the DNA would be sucked through the pores.
As can be seen from Video 1. "Oxford Nanopore MinION software channels pannel on Mac." the software tells us which pores are still working.
Figure 5.
Oxford Nanopore MinION connected to a Mac via USB.
Source.
Video 1.
Oxford Nanopore MinION software channels pannel on Mac.
Source.
Pores go bad sooner or later randomly, until there are none left, at which point we can stop the process and throw the flow cell away.
48 hours was expected to be a reasonable time until all pores went bad, and so we called it a day, and waited for an email from the PuntSeq team telling us how things went.
We reached a yield of 16 billion base pairs out of the 30Gbp nominal maximum, which the bio people said was not bad.
Because it is considered the less interesting step, and because it takes quite some time, this step was done by the event organizers between the two event days, so participants did not get to take many photos.
PCR protocols are very standard it seems, all that biologists need to know to reproduce is the time and temperature of each step.
We did 35 cycles of:
Figure 1.
Marshal Scientific MJ Research PTC-200 Thermal Cycler.
Source.
We added PCR primers for regions that surround the 16S DNA. The primers are just bought from a vendor, and we used well known regions are called 27F and 1492R. Here is a paper that analyzes other choices: academic.oup.com/femsle/article/221/2/299/630719 (archive) "Evaluation of primers and PCR conditions for the analysis of 16S rRNA genes from a natural environment" by Yuichi Hongoh, Hiroe Yuzawa, Moriya Ohkuma, Toshiaki Kudo (2003)
One cool thing about the PCR is that we can also add a known barcode at the end of each primer as shown at Code 1. "PCR diagram".
This means that we bought a few different versions of our 27F/1492R primers, each with a different small DNA tag attached directly to them in addition to the matching sequence.
This way, we were able to:
  • use a different barcode for samples collected from different locations. This means we
    • did PCR separately for each one of them
    • for each PCR run, used a different set of primers, each with a different tag
    • the primer is still able to attach, and then the tag just gets amplified with the rest of everything!
  • sequence them all in one go
  • then just from the sequencing output the barcode to determine where each sequence came from!
Input: Bacterial DNA (a little bit)
... --- 27S --- 16S --- 1492R --- ...

|||
|||
vvv

Output: PCR output (a lot of)
Barcode --- 27S --- 16S --- 1492R
Code 1.
PCR diagram
.
Finally, after purification, we used the Qiagen QIAquick PCR Purification Kit protocol to purify the generated from unwanted PCR byproducts.
Biology experiments are hard, and so they go wrong, a lot.
For this reason, it is wise to verify that certain steps are correct whenever possible.
And so this is the first thing we did on the second day!
Gel electrophoresis separates molecules by their charge-to-mass ratio. It is one of those ultra common lab procedures!
This allows us to determine how long are the DNA fragments present in our solution.
Since we know that we amplified the 16S regions which we know the rough size of (there might be a bit of variability across species, but not that much), we were expecting to see a big band at that size.
And that is exactly what we saw!
First we had to prepare the gel, put the gel comb, and pipette the samples into wells present in the gel:
Figure 1.
Gel electrophoresis insert comb.
Source.
Figure 2.
Gel electrophoresis top view with wells visible.
Source.
Figure 3.
Gel electrophoresis pipette sample into wells.
Source.
To see the DNA, we added ethidium bromide to the samples, which is a substance that that both binds to DNA and is fluorescent.
Because it interacts heavily with DNA, ethidium bromide is a mutagen, and the biology people sure did treat the dedicated electrophoresis bench area with respect! Figure 4. "Gel electrophoresis dedicated bench area to prevent ethidium bromide contamination.".
Figure 4.
Gel electrophoresis dedicated bench area to prevent ethidium bromide contamination.
Source.
Figure 5.
Gel electrophoresis dedicated waste bin for centrifuge tubes and pipette tips contaminated with ethidium bromide.
Source.
The UV transilluminator we used to shoot UV light into the gel was the Fisher Scientific UVP LM-26E Benchtop 2UV Transilluminator. The fluorescent substance then emitted a light we can see.
As barely seen at Figure 8. "Fischer Scientific UVP LM-26E Benchtop 2UV Transilluminator illuminated gel." due to bad photo quality due to lack of light, there is one strong green line, which compared to the ladder matches our expected 16S length. What we saw it with the naked eyes was very clear however.
Figure 6.
Fischer Scientific UVP LM-26E Benchtop 2UV Transilluminator
. Source.
Figure 7.
Fischer Scientific UVP LM-26E Benchtop 2UV Transilluminator loading gel.
Source.
Figure 8.
Fischer Scientific UVP LM-26E Benchtop 2UV Transilluminator illuminated gel.
Source.
After filtration, all DNA should present in the filter, so we cut the paper up with scissors and put the pieces into an Eppendorf: Video 1. "Cutting vacuum pump filter and placing it in Eppendorf".
Video 1.
Cutting vacuum pump filter and placing it in Eppendorf
. Source.
Now that we had the DNA in Eppendorfs, we were ready to continue the purification in a simpler and more standardized lab pipeline fashion.
First we added some small specialized beads and chemicals to the water and shook them Eppendorfs hard in a Scientific Industries Inc. Vortex-Genie 2 machine to break the cell and free the DNA.
Once that was done, we added several reagents which split the solution into two phases: one containing the DNA and the other not. We would then pipette the phase with the DNA out to the next Eppendorf, and continue the process.
In one step for example, the DNA was present as a white precipitate at the bottom of the tube, and we threw away the supernatant liquid: Figure 1. "White precipitate formed with Qiagen DNeasy PowerWater Kit".
Figure 1.
White precipitate formed with Qiagen DNeasy PowerWater Kit
. Source.
At various stages, centrifuging was also necessary. Much like the previous vacuum pump step, this adds extra gravity to speed up the separation of phases with different molecular masses.
In our case, we used a VWR Micro Star 17 microcentrifuge for those steps:
Figure 2.
VWR Micro Star 17 microcentrifuge.
Source.
Figure 3.
VWR Micro Star 17 microcentrifuge loading.
Source.
Then, when we had finally finished all the purification steps, we measured the quantity of DNA with a Biochrom SimpliNano spectrophotometer to check that the purification went well:
Figure 4.
Biochrom SimpliNano spectrophotometer loading sample.
Source.
Figure 5.
Biochrom SimpliNano spectrophotometer result readout.
Source.
And because the readings were good, we put it in our -20 C fridge to preserve it until the second day of the workshop and called it a day:
Figure 6.
Minus 20 fridge storing samples.
Source.
One cool thing we did in this procedure was to use magnetic separation with magnetic beads to further concentrate the DNA: Figure 1. "GE MagRack 6 pipetting.".
The beads are coated to stick to the DNA, which allows us to easily extract the DNA from the rest of the solution. This is cool, but bio people are borderline obsessed by those beads! Go figure!
Figure 1.
GE MagRack 6 pipetting.
Source.
Figure 2.
GE MagRack 6 eppendorf with magnetic beads mounted.
Source.
Here some of the steps required a bit more of vortexing for mixing the reagents, and for this we used the VELP Scientifica WIZARD IR Infrared Vortex Mixer which appears to be quicker to use and not as strong as the Vortex Genie 2 previously used to break up the cells:
Figure 3.
VELP Scientifica WIZARD IR Infrared Vortex Mixer running.
Source.
After all that was done, the DNA of the several 400 ml water bottles and hours of hard purification labour were contained in one single Eppendorf!
Parasites tend to have smaller DNAs Updated +Created
If you live in the relatively food abundant environment of another cell, then you don't have to be able to digest every single food source in existence, of defend against a wide range of predators.
And likely you also want to be as small as possible to evade the host's immune system.
Power, Sex, Suicide by Nick Lane (2006) section "Gene loss as an evolutionary trajectory" puts it well:
One of the most extreme examples of gene loss is Rickettsia prowazekii, the cause of typhus. [...] Over evolutionary time Rickettsia has lost most of its genes, and now has a mere  protein-coding genes left. [...] Rickettsia is a tiny bacterium, almost as small as a virus, which lives as a parasite inside other cells. It is so well adapted to this lifestyle that it can no longer survive outside its host cells. [...] It was able to lose most of its genes in this way simply because they were not needed: life inside other cells, if you can survive there at all, is a spoonfed existence.
and also section "How to lose the cell wall without dying" page 184 has some related mentions:
While many types of bacteria do lose their cell wall during parts of their life cycle only two groups of prokaryotes have succeeded in losing their cell walls permanently, yet lived to tell the tale. It's interesting to consider the extenuating circumstances that permitted them to do so.
[...]
One group, the Mycoplasma, comprises mostly parasites, many of which live inside other cells. Mycoplasma cells are tiny, with very small genomes. M. genitalium, discovered in 1981, has the smallest known genome of any bacterial cell, encoding fewer than 500 genes. M. genitalium, discovered in 1981, has the smallest known genome of any bacterial cell, encoding fewer than 500 genes. [...] Like Rickettsia, Mycoplasma have lost virtually all the genes required for making nucleotides, amino acids, and so forth.
Paris Updated +Created
Ciro Santilli lived in Paris for a few years between 2013 and 2016, and he can confirm the uncontroversial fact that "Paris is Magic".
Not just one type of magic though. Every quarter in Paris has its own unique personality that sets it apart and gives it a different mood.
Ciro knows Paris not from its historical facts, but from the raw feeling of endless walks through its streets in different times of the year. Ciro is a walker.
Maybe one day Ciro will expand this section to try and convey into words his feelings of love for the city, but maybe the effort would be pointless. Maybe such feelings can only be felt by other free-roaming walker souls living in the city, and that is both beautiful and a shame.
Ciro had written the following in the past before he lived in smaller cities, started cycling and joined the Street reclamation movement he thought:
Paris is a friendly city to walkers, as it is not too large, and does not have too many extremely busy roads, you can basically cross all of it on foot.
Perhaps compared to São Paulo City, which is what he knew before that was true. But no, his standards have improved since. Paris has way too many cars. The noise of internal combustion engine vehicles is extremely annoying. And because there are too many personal vehicles, cars have to horn a lot to fight for space. Fuck cars. Paris has been making a big cycling push in the early 2020's, and that is great. But it is still far, far from good.
Path integral formulation Updated +Created
This one might actually be understandable! It is what Richard Feynman starts to explain at: Richard Feynman Quantum Electrodynamics Lecture at University of Auckland (1979).
The difficulty is then proving that the total probability remains at 1, and maybe causality is hard too.
The path integral formulation can be seen as a generalization of the double-slit experiment to infinitely many slits.
Feynman first stared working it out for non-relativistic quantum mechanics, with the relativistic goal in mind, and only later on he attained the relativistic goal.
TODO why intuitively did he take that approach? Likely is makes it easier to add special relativity.
This approach more directly suggests the idea that quantum particles take all possible paths.
Path to AGI Updated +Created
There are two main ways to try and reach AGI:
Which one of them to take is of of the most important technological questions of humanity according to Ciro Santilli
There is also an intermediate area of research/engineering where people try to first simulate the robot and its world realistically, use the simulation for training, and then transfer the simulated training to real robots, see e.g.: realistic robotics simulation.
Pauli-X gate Updated +Created
The quantum NOT gate swaps the state of and , i.e. it maps:
As a result, this gate also inverts the probability of measuring 0 or 1, e.g.
Equation 2.
Quantum NOT gate matrix
.
Figure 1.
Quantum NOT gate symbol
. Source.
Peano existence theorem Updated +Created
Peptidoglycan Updated +Created
From the Wikipedia image we can see clearly the polymer structure formed: it is a mesh with:
Figure 1.
Peptidoglycan polymer structure
. Source.
Pernosco Updated +Created
Proprietary extension to Mozilla rr by rr lead coder Robert O'Callahan et. al, started in 2016 after he quit Mozilla.
Physics education needs more focus on understanding experiments and their history Updated +Created
This is the only way to truly understand and appreciate the subject.
Understanding the experiments gets intimately entangled with basically learning the history of physics, which is extremely beneficial as also highlighted by Ron Maimon, related: there is value in tutorials written by early pioneers of the field.
"How we know" is a basically more fundamental point than "what we know" in the natural sciences.
In the Surely You're Joking, Mr. Feynman chapter O Americano, Outra Vez! Richard Feynman describes his experience teaching in Brazil in the early 1950s, and how everything was memorized, without any explanation of the experiments or that the theory has some relationship to the real world!
Although things have improved considerably since in Brazil, Ciro still feels that some areas of physics are still taught without enough experiments described upfront. Notably, ironically, quantum field theory, which is where Feynman himself worked.
Feynman gave huge importance to understanding and explaining experiments, as can also be seen on Richard Feynman Quantum Electrodynamics Lecture at University of Auckland (1979).
Video 1.
'Making' - the best way of learning science and technology by Manish Jain (2018)
Source.
Physics research institute Updated +Created
Pineapple jelly with cream Updated +Created
December 2023: www.tudogostoso.com.br/receita/81176-gelatina-de-abacaxi-com-creme-de-leite.html Terribly explained recipe! Used 5 spoons of sugar rather than 10, and a 300ml cup of double cream. Turned out OK, except that the cream condensed all on top, and assumed the same coarse texture as when you do a fatty beef and let it cool, so not so nice,
Maybe this would be more successful: receitas.globo.com/tipos-de-prato/doces-e-sobremesas/gelatina-de-abacaxi-4e64345bddf17214b4003e71.ghtml They also use condensed milk, and beat the cream with the jelly, so it might mix better? It didn't really.
June 2024: Now going for:
  • 4 cups of water
  • 1 spoon of sugar
  • just drop 150 ml double cream on top after jelly and mix with spoon since anything else was pointless to get mixture
For some reason it became too liquid this time, the jelly didn't work very well. Not sure why. The pineapple was a bit large.
Pinto bean Updated +Created
This seems to be the "brown Brazilian bean" that many Brazilians eat every day.
Edit: after buying it, not 100% sure. This one felt smaller than what Ciro had in Brazil, borlotti beans might be closer. Pinto beans are smaller, and creamier, and have softer peel, possibly produced less natural gas.
2021-04: second try.
2021-03: did for first time, started with same procedure as borlotti beans 2021-03. Maybe 1h30 is too much. Outcome was still very good.

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