PCR by Ciro Santilli 35 Updated +Created
More generic PCR information at: Section "Polymerase chain reaction".
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:
  • 94˚C for 30 seconds
  • 60˚C for 30 seconds
  • 72˚C for 45 seconds
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.
Private university by Ciro Santilli 35 Updated +Created
Undergraduate education by Ciro Santilli 35 Updated +Created
University entry exam by Ciro Santilli 35 Updated +Created
These are the most evil examinations society has.
They mean that until you are 18, you have to study a bunch of generic crap you hate just to get into university. Rather than studying whatever it is that you truly love to become a God at it as fast as possible and have any chance of advancing the field.
And then, if you decide that you want to change, which is not unlikely since you haven't really try to study what you signed up for before then, it can be very hard and time consuming, leading to a bunch of adults with useless degress they will never use at work.
With the invention of the Internet, all teaching material can be free and open source. Only laboratory space has any cost (besides the opportunity cost of participation in actual projects in a research team).
Per-country list: en.wikipedia.org/wiki/List_of_admission_tests_to_colleges_and_universities
Equipment used by Ciro Santilli 35 Updated +Created
Australian university by Ciro Santilli 35 Updated +Created
Public Ivy by Ciro Santilli 35 Updated +Created
American university by Ciro Santilli 35 Updated +Created
Harvard University by Ciro Santilli 35 Updated +Created
De novo DNA synthesis by Ciro Santilli 35 Updated +Created
As of 2018, Ciro Santilli believes that this could be the next big thing in biology technology.
"De novo" means "starting from scratch", that is: you type the desired sequence into a computer, and the synthesize it.
The "de novo" part is important, because it distinguishes this from the already well solved problem of duplicating DNA from an existing DNA template, which is what all our cells do daily, and which can already be done very efficiently in vitro with polymerase chain reaction.
Many startup companies are attempting to create more efficient de novo synthesis methods:
Notably, the dream of most of those companies is to have a machine that sits on a lab bench, which synthesises whatever you want.
TODO current de novo synthesis costs/time to delivery after ordering a custom sequence.
The initial main applications are likely going to be:
but the real pipe dream is building and bootstraping entire artificial chromosomes
News coverage:
Video 1.
Nuclera eDNA enzymatic de novo DNA synthesis explanatory animation (2021)
Source. The video shows nicely how Nuclera's enzymatic DNA synthesis works:
Artificial chromosome by Ciro Santilli 35 Updated +Created
Genome Project-Write by Ciro Santilli 35 Updated +Created
Transcription (biology) by Ciro Santilli 35 Updated +Created
Post-transcriptional modification by Ciro Santilli 35 Updated +Created
Nucleotide by Ciro Santilli 35 Updated +Created
Nucleobase by Ciro Santilli 35 Updated +Created
Cytosine by Ciro Santilli 35 Updated +Created
Start codon by Ciro Santilli 35 Updated +Created
Pinned article: ourbigbook/introduction-to-the-ourbigbook-project
Welcome to the OurBigBook Project! Our goal is to create the perfect publishing platform for STEM subjects, and get university-level students to write the best free STEM tutorials ever.
Everyone is welcome to create an account and play with the site: ourbigbook.com/go/register. We belive that students themselves can write amazing tutorials, but teachers are welcome too. You can write about anything you want, it doesn't have to be STEM or even educational. Silly test content is very welcome and you won't be penalized in any way. Just keep it legal!
Video 1.
Intro to OurBigBook
. Source.
We have two killer features:
  1. topics: topics group articles by different users with the same title, e.g. here is the topic for the "Fundamental Theorem of Calculus" ourbigbook.com/go/topic/fundamental-theorem-of-calculus
    Articles of different users are sorted by upvote within each article page. This feature is a bit like:
    • a Wikipedia where each user can have their own version of each article
    • a Q&A website like Stack Overflow, where multiple people can give their views on a given topic, and the best ones are sorted by upvote. Except you don't need to wait for someone to ask first, and any topic goes, no matter how narrow or broad
    This feature makes it possible for readers to find better explanations of any topic created by other writers. And it allows writers to create an explanation in a place that readers might actually find it.
    Figure 1.
    Screenshot of the "Derivative" topic page
    . View it live at: ourbigbook.com/go/topic/derivative
    Video 2.
    OurBigBook Web topics demo
    . Source.
  2. local editing: you can store all your personal knowledge base content locally in a plaintext markup format that can be edited locally and published either:
    • to OurBigBook.com to get awesome multi-user features like topics and likes
    • as HTML files to a static website, which you can host yourself for free on many external providers like GitHub Pages, and remain in full control
    This way you can be sure that even if OurBigBook.com were to go down one day (which we have no plans to do as it is quite cheap to host!), your content will still be perfectly readable as a static site.
    Figure 5. . You can also edit articles on the Web editor without installing anything locally.
    Video 3.
    Edit locally and publish demo
    . Source. This shows editing OurBigBook Markup and publishing it using the Visual Studio Code extension.
    Video 4.
    OurBigBook Visual Studio Code extension editing and navigation demo
    . Source.
  3. https://raw.githubusercontent.com/ourbigbook/ourbigbook-media/master/feature/x/hilbert-space-arrow.png
  4. Infinitely deep tables of contents:
    Figure 6.
    Dynamic article tree with infinitely deep table of contents
    .
    Descendant pages can also show up as toplevel e.g.: ourbigbook.com/cirosantilli/chordate-subclade
All our software is open source and hosted at: github.com/ourbigbook/ourbigbook
Further documentation can be found at: docs.ourbigbook.com
Feel free to reach our to us for any help or suggestions: docs.ourbigbook.com/#contact