How I Defeated My Arch Nemesis

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BL21 E. coli are by far the most obnoxious bacteria genetic engineers usually work with for 1 simple reason. They are maddeningly difficult to modify and load with new DNA. This wouldn't be an issue if they weren't also one of the best types for producing proteins en mass, and as such have application in everything from pharmaceutical production, to producing ingredients for foods. But after a few years of tinkering with it I finally found a protocol that reliably fixes this irritating issue, and makes it easy to transform them reliably.

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[alexbenavidez]

"You can fart DNA in their general direction and they'll take it in"
That's a great fucking line of a biochemist.

[ExplosionsAndFire]

Science is temporary, grudges are forever xx

[waterunderthebridge]

6:06 It still surprises me how a lot of times in biology/medicine it’s just

“We don’t care _how_ it works exactly. If it works consistently, we don’t question the process. Don’t anger the science gods with your impious insolence!”

[KateeAngel]

"Everything in biology is yellow".
Me, a cyanobacteriologist: "Cool story, bro!"

[Pyriphlegeton]

4:26
"Sort of like Bioshock but less shooting Spiders and Lightning"
*...for now.*

[bryby]

I'm a molecular biologist in an academic lab, and dude, I'm so thankful for your videos. It's easy to forget how cool what we do is, and instead just treat it like a grind. Thanks for making these videos, and thanks for sharing your protocol. It's refreshing

[ace-kzid]

I might not understand this stuff at all but it is super intresting

[stijn]

This is one of the things I like about you. When you discover something, instead of patenting it or trying to sell a method you came up with to a company, you make it available to the public and I appreciate that. Thank you

[moonface]

Obligatory "yellow chem bad"

[afroninjaman]

The superstition comment got me. I've always said genetics is half black magic. In my capstone project lab the grad students had special P10 pipettes labelled "Golden Child" and "Vampire" because respectively the former made reactions generally and PCR specifically work like magic, and the latter drained the life out of your experiments.

[VincentGroenewold]

I worked in science and doing things like this for over 10 years and 1 thing I learned is how everyone always copies protocols to the T. It may make sense of course, but it's never really a thing people think hard about it seems. I once started research on an entire new species for our work, got a protocol from another lab full of alternative approaches and no real explanation as to why. Turned out they had 1 step I recognized to likely be extremely in-efficient. So I just did it my way and presto, totally worked. That was when I concluded this probably happens a lot in labs. That protocol was in use for years as well.

[NicRobertsNerd]

What an amazing thing to be living in a time when credible science procedural tutorials are available for free on YouTube. Thank you so much for posting this.

[karigucio]

the advert i got before this vid was about getting rid of ecoli from the water. Quite on point

[Quroe_]

You've made bioengineering somewhat understandable to me, a mechanical engineer. I hope I get to collaborate and effectively communicate with a bioengineer well enough later in my life and make something cool because of you.

[janrace]

3:35 so THAT's why the university of biology in my country keeps a herd of sheep!

[zenzetsu]

Me: subscribes to The Thought Emporium for his physics videos with little knowledge or interest in biology
The Thought Emporium: releases biology video
Me watching the biology video anyway: I like your funny words magic man

[-w-.]

Now that E&F is taking a break, I'm glad that someone decided to take his place.

[NachozMan]

I've been learning Micro Biology for college this semester and I'm starting to understand what you talk about a bit more in your videos now! Really cool!

[lucashuttebraucker]

Hey there, just a few tipps:
When you prepare your cells fresh every time you have a big margin of error.
I would suggest to prepare competent cells in advance supplement the buffer with 15% glycerol and freeze them. There are lots of viable protocols available. Important is just to work on ice the whole time, and keep the cells always cold.

Best at -80c but -20 is sufficient for a week or two.
Also I recommend to transform one prepared aliqout with a standardized plasmid and calculate the theoretical transformation efficiency.
Then you can transform cells within 2 h.

Also for plating the cells I personally prepare always 2 plates, one with 100 ul of the cells in soc media and one with the rest one with the rest.

Finally if you can't or don't want to prepare cells ahead, you can use TSS transformation, it is fast, simple and idiot proof. You can use e coil directly from the plate. Just the efficiency is not that great.

[blackychan]

I understood almost nothing but still enjoyed every minute