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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
This process used a Marshal Scientific MJ Research PTC-200 Thermal Cycler:
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
Finally, after purification, we used the Qiagen QIAquick PCR Purification Kit protocol to purify the generated from unwanted PCR byproducts.
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).
"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
- polymerase chain reaction primers (determine which region will be amplified
- creating a custom sequence to be inserted in a plasmid, i.e. artificial gene synthesis
News coverage:
- 2023-03 twitter.com/sethbannon/status/1633848116154880001
AnsaBio created the world's longest DNA oligo produced using de novo synthesis! 1,005 bases! 99.9% stepwise yield
- 2020-10-05 www.nature.com/articles/s41587-020-0695-9 "Enzymatic DNA synthesis enters new phase"
Using de novo DNA synthesis to synthesize entire Chromosomes.
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!
We have two killer features:
- 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-calculusArticles 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. - 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: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.
- 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
- Internal cross file references done right:
- Infinitely deep tables of contents:
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