Extracting Spider/Bacteria DNA Using Columns - Spider Silk Step 1
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- Опубликовано: 6 сен 2024
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DNA extraction is easily one of the most common procedures in a genetics laboratory. Maybe we want to isolate a new gene from some organism, or we just want more of a plasmid we designed. No matter what chances are you'll use a column based method.
Today we explore the basics of column DNA extraction and look at 2 different kits for 2 different DNA sources and explore why they're different.
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![Megan Thee Stallion - Neva Play (feat. RM) [Official Video]](http://i.ytimg.com/vi/TpYTyAaTRts/mqdefault.jpg)
We are insanely lucky that this person is a nice mad scientist.
as far as I've seen madness is a prerequisite to work in any field of science
atleast for the entirety of my classmates and teachers
@@sachiel197 well you have to be some sort of mad to work on a very tiny detail for years at a time that the average person doesn't know or care about.
“So I made some chloroform”
Jai Carlson like Dr. Doofenschmirtz, despite Dr. D’s attempts otherwise, but without the musical sequences and marsupial nemesis.
We don’t know that for sure.
10:15 I think the spider is probably not well conserved, thus the degraded DNA. It might be that the ethanol did not get into the spider well enough due to the strong cuticular. I worked with lot's of insects for DNA extraction, and as long as they are conserved in 96% ethanol (and the ethanol is exchanged with fresh 96% after a few hours) the specimens give good DNA. The replacement of ethanol can be important as the specimens contain water that dilutes the ethanol. A ethanol concentration of 70-80% might lead to DNA degradation over time. Not sure here, since I would think most enzymes would be denatured by the ethanol, so maybe it's just "normal" tissue / DNA degradation? Not sure here what exactly drives DNA degradation, but 96% ethanol should not lead to DNA degradation. Great video as always = )
Spider silk and yeast. The most ambitious crossover of 2019
Drunk spooderman
'Cells are basically living bubbles of oil.' Can I write that in my exam?
water bubbles coated with an oily film
Cells: *are basically living bubbles of oil*
USA: *FREE REAL STATE LOCATED*
@@clausroquefort9545 filled with a bunch of stuff and a powerhouse
@@psun256*powerhouses
Met his dad he is my new neighbours. After talking for 4 minutes he goes: "my kid is a genius and doe youtube for a living, I'm like yeah sure"... then this happened....
Next month: How to make your own antivenom if necessary!
2 months later: "Pharmaceutical companies made me take my video down"
@@wumbology8421hahaha lol
Haha. Ha! :)
You forgot "from dog Feces"
Don’t tempt him now
This is going to be a great series.
BTW, right now I'm recreating your kombucha leather project and it's doing great, especially considering it's winter
also I found out you can kind of get the kombucha to grow on a specific mesh by accidentally having the gauze I used to cover it touch the water. think beyond leather: not only can we make it florescent using stuff like you do here, we can have steel - reinforced celluloze fabric, tailored specifically to the thickness and shape we need and requiring only a bit of fructose and perhaps tea (I'm currently testing whether you actually need the tea).
Gonna be revisiting this soon actually. Found a better way to make the sheets that doesn't require being super careful around the tanks. Just grow the scoby as thick as you can, blend it, add a bit of afresh tea and then "cast" it into whatever form you want. You can suspend mesh at this step to radically increase strength too. As it regrows it knits all the shards back together into a strong final piece that's perfect every time
ממש מגניב
Would using coffee make the process faster? (Just a child level idea)
אתה עושה את הניסוי במסגרת מסוימת או לבד
I'm wondering if you can use yard waste such as fallen leaves as a tea replacement.
“...so i made some chloroform...” you know, just casually
I mean, it's not that hard. You can make it with average household ingredients.
schnee It’s like bleach and some other household cleaner, right?
Storyman Bleach an Acetone (nailpolish remover)
Its super easy to make.
The plasmid kit shouldn't work for genomic DNA of any kind. When you "crash" out the proteins with the third buffer. The genomic DNA should be in tact enough that the genomic DNA itself will "crash out". Since the plasmid DNA is smaller it will stay in solution. So just heads up that that kit shouldn't work for yeast or bacterial genomic DNA.
A couple of things I ran into during column-based DNA extraction:
1) Columns tend not to release DNA fragments longer than around 15Kbp, so if the protocol was performed too gently, lots of DNA can remain in the column. Bead-based kits, either magnetic or silica bead-based (the majority of MPbio DNA extraction kits are the latter) will work better for longer fragments. However, the MPbio silica bead kits are prone to column clogging if the samples have a high protein content (isolating DNA from earthworms turns these kits into a labor-intensive plumber simulator).
2) The final DNA yield with any silica-based kit can be improved by heating up a column with elution buffer for around 5 minutes in a 45-50 centigrade heat block before a spindown. Actually, in my experience, it's essential for getting a sensible amount of DNA out of DNA-lean samples like sand or permafrost soil samples.
I learned basic programming skills and how to build robots of questionable utility entirely because of the Web, and gained a hobbyist's competency in 3D modelling and CNC macguffin building/operation almost solely thanks to RUclips and Reddit posts. I still find it just plain amazing to see you confidently talking about genetic engineering and doing the work. You bet I clicked the bell.
I am so glad I found your stuff!! So cool!
Hey, I work in an arachnology lab. If you are wanting just a couple of southern widows (L. mactans), you are welcome to the couple of live ones I keep. If you need larger quantities (like 100 or more), I'll have to collect them closer to the fall when they are more plentiful.
Really enjoying this series. Thanks for sharing.
By the way, the main goal of adding Proteinase K is to get rid of DNAses and avoid excessive DNA cleavage during the low-EDTA stages with proteins still present in the solution.
In my Lab we use the spectrometer just to check for impurities. The 260nm absorbtion rate tells you about the basic concentration while the 260/280nm ratio is a hint for protein contamination, a ratio above 1.5 - 2.0 would be to contaminated (depending on how strict and clean you want /can work).
Many spiders were harmed in the making of this video
But its okay....
BECAUSE IT'S FOR SCIENCE :D
@@User-nu6km the difference between science and messing about is writing down what's happening.
@Luca Stronks yea, and venomous ones at that
And just spiders. Bulbous spiders
Lukarjo you aren’t phobic are you?
Phobia is not exactly reasonable unfortunately.
Lukajro lol u legitimately can not fight phobia it is an instinct
I talk from experience btw
you can get the purity by A260/280 in spectrophotometer
loving this series
Really like your videos man they're excellent! 2 questions: 1 - I would love to build a small lab myself but it's prohibitively expensive for me... just a specrophotometer alone is a couple grand at least. Do you own all that equipment, and which suppliers would you recommend are good value for what they produce? 2 - What do you intend on doing with the spider silk if you manage to mass produce it using yeast? I finished my undergrad in biochem 4 years ago but have since moved over completely to tech, so would love to revisit biology/chem perhaps as a hobby on the side once funds allow for it
Mate, do you even sleep sometimes ?
:D
Awesome content, I don't think I know a youtuber that is working on so many projects from so many categories :D
Your videos are so good! Very technical, but also easy to digest. And you show all the hands on aspects as well!
It was kinda sad following your disappointments on Instagram.
You could have used a push transition for the scene where you explain every single subprocess around 2minutes and it would look much better.
It's science. It's all disappointment all the time... and then it works and you buy champagne. Also this is an incredibly difficult project. Like PhD level at least. And we've only been at it for a couple months while I'm still building the lab. I'd have been surprised if it worked on the first go and went into this expecting a lot of failure. But it makes the moment when things do work a lot better. I've got 2 fresh spiders en route and new primers coming in the mail. Should hopefully sort this out.
Nice video on DNA extraction!
I have some suggestions: When using tissue samples, they should either be as fresh as possible or frozen using LN2 or a -80C refridgerator.
You might get around the tedious DNA extraction by using the same principle as in colony PCRs with bacteria. Just poke your (fresh) spider, dip the needle in a PCR solution (ontaining the primers for the silk gene) and start the PCR.
Or do you actually need the genomic DNA in large quantities?
We tried that actually. Didn't work. But could be the spider quality is too low. When I get fresher ones I'll try it again.
Like he was saying, spiders (and other complex eukaryotes) often produce large amounts of PCR inhibitors in their cells. I don't think a colony-PCR approach would work.
To really preserve DNA in samples include a chelator like EDTA. DNAses almost always require Mg to work, RNAses are more difficult.
Funnily enough.
This semester my BioLab is working on a project where we catch spiders, extract their DNA, and sequence it in order to create phylogenetic trees.
The day before yesterday we caught like 50 spiders from a few species at a local park.
No black widows but tons of some more common species.
Unless there's something different to black widow spider silk, I recommend you find out what the most common species is in your area and go out and catch a few.
That way the DNA isn't all digested by the time of the extraction.
Why not try your PCR reaction on the degraded spider DNA? Your sequence might still be intact
We did. Didn't work unfortunately. In fact we tried like 3 variants of that, and 2 sets of primers but no dice
@@thethoughtemporium Doesn't it look odd? As far as I remember the sequence you are trying to extract isn't that long, i thought there will be enough undamaged material, given sensitivity of PCR.
Assuming that he was using a 100 base pair ladder, it looked like most of the spider DNA was less than 100 bp (the lowest rung of the ladder). His gene of interest is likely much larger than 100 bp, so I think he'd have a tough time getting the amount of amplification required to insert his sequence into a plasmid...
I really don't know much but this is fun and maybe in future i will study this field of science
Resin columns are generally used for alkaline pH
You can also store your spiders for a fairly long time in CTAB. This is often what biologists do while collecting specimens in the field.
Lol got an acestry Advertisement for this video
unknown bc me too - hope I don‘t have to be shredded like the spiders to get my results
@@ocng It's just a few legs. Don't worry.
I wonder what would happen if you sent the spider DNA to ancestry
Great video, I am learning a lot. Couldn't you just order an oligo of the spider gene for silk and use that in your experiment with the yeast?
Interesting!!!😎👍 How much did the lab equipment cost?
Lots
Just a possible idea. Due to Spiders having specialized cells that create this spider silk, those cells create the silk in high quantities. Your Ecoli is not optimized for spider silk production. You should try and simplify your plasmid or use a high copy number plasmid, as well as extracting 2-3 spiders at a time.
Can you explain to me why DNA is negatively charged? It makes sense to me that if DNA is an acid (Desoxiribonucleic Acid) it should be positively charged...
You and nile red are my favorite youtubers
Max Turner their videos make my head hurt sometimes but in a GOOD way, i learn so much from their videos
Might be a bit late, but I live in northern texas and there are black widows EVERYWHERE here. If you want to get in touch I could ship a couple live ones over.
I study biology at university and this is way more intresting than most things we see!!!
Also i think you could use a jack , same jacks they use in chemistry , they are quite inexpensive even 3d printable , and then you no longer need to stack stuff to work on eye level ;)
Also couldn't you get live spiders from like a local spider keeper?
Oh lol we did basically what he's doing in the first bit in school, where we extracted DNA from tomatoes, but of course we didn't have any fancy kits or a centrifuge
One question I have always had is there companies that will modify DNA for you But how do they modify something so small Optical tweezers could probably move it
I was a cook for 4 years while in college, now im going to grad school for biochemistry 😮
Trying to extract from fly larvae by just using isopropanol precipitate and i have no bands 🥹
If you want to capture the DNA without letting enzymes get a chance to damage it look into obtaining some liquid Nitrogen and cyro freeze the live spider before harvesting the tissue section you process for DNA.
Tell me the names of the kit please, wanna genetically modify a jumping spider in the future
Hi, has there been an update on this project? If you're still looking to procure spiders would redback spiders work? There's tons of people in Australia who deal with infestations of them, leokimvideo for example. I'm sure if you reached out on social media you could find someone willing to trap and ship you some fresh spiders. Right now it's the summer over there, perfect time to round up some fresh ones.
This man could work for oscorp one day
I like the magnets on the centrifuge!
Happy to see you got a sponsor
>Plugs Nigel's channel
Canadians supporting Canadians. Warms my heart.
09:30
Absorbance at 230nm is too high and there isn't a peak at 260nm, you have a peak at 270-280nm (organic stuff) . The important thing is A260/A280, in your case it result 0,56 -> no DNA
Where is the rest of this, I was so happy this is old so it would be finished but it is not apparently :/
Biology is hard. I'm still working on it.
@@thethoughtemporium Still glad to hear it is ongoing :) You really inspired me about genetic engineering
I'm watching this sitting like three feet away from my phone. Spiders terrify me.
3:03 So the dna gets through the filter when it is not charged?
Heck, I have HUNDREDS of rather large black widows (and several other spiders of the Theridiidae family) just hanging round my house and woodpile. Shall I box a few of em up on ice? lol
Depends. What species are they? If you're from florida or the east coast, then no. But if you're from the south west (arizona and such) then maybe.
Is there a gene that gives the spiders their scopulae en how can you do a extraction on them
Why are you extracting genomic DNA from the spiders instead of extracting RNA and reverse transcribing into cDNA? Does the gene encoding spider silk have introns? If so will the yeast be able to properly splice the resulting transcript properly once you ligate the spider silk gene into a plasmid and transform it into the yeast?
No introns. Just solid code
Cool! Thanks for the quick reply!
What book is great for growing /changing bacteria?
I have a design for a perfect webshooter already:>
Anybody else noticed the centrifuge tubes rotating by themselves at 5:06 ?????
Your channel need more subs, amazing content
you could breed spiders at home. once you get some they're pretty cheap to keep alive and don't take much of space
Too bad you are not in the USA... We have some invasive Latrodectus geometricus that are incredibly abundant and would be easy to source from Southern California with zero qualms about reducing native spider populations. If you know what specifics would need to be satisfied on the legal front that may be a viable option for you to look into...
Qny one have a link on a kit I could use to make two mushrooms in to a hybrid
This may be well beyond the capabilities of your lab, and I could be wrong, but wouldn't it be better if you splice the spider's silk genes into the common silk worm instead of into yeast? Theoretically at least.
You had me at "barbecue sauce"
I'm looking forward to see the next video.
I saw this was old so I was happy because I thought part 2 would be out :( did it fail and you stoped working on it or is it just that biology takes a long time??
Biology takes a long time. I think I've finally made silk, so will be putting out an update next month
The Thought Emporium i love how you are so interactive with viewers I hope you won’t have anymore road blocks with covid
@@thethoughtemporiumupdate
I live in the desert and had a black widow hatch an egg in my lab...plenty of spider dna here for ya.
Why and How are the tubes rotating in 5:11?
Don't know if you know any body that lives in Nevada, but I used to live there and I know a spot that literally had thousands of black widows. I was a kid though,I used to catch bugs to feed to them.
THANK YOU.
Will you consider being a guest on my show, I find your research fascinating.
It's a great initiative
he often says "DNA circles" ... that confuses me, I thought DNA is a single string... yet he makes it seem like there are tons of different DNA circles next to each other?
We strive to understand microorganisms and we once were microorganisms, kind of interesting.
How did you make Chloroform? Also are you doing this out of your home or at work?
Is black widow silk really the best? If not, why use those? - Just use any spider. It seems crazy to me someone would buy spiders, with the amp supply everybody already has...
one of the few species that produce exceptionally strong silk, but hasn't been patented so I can release it open source
@@thethoughtemporium Oh, whow! Thank you, AND Than you for everyone that will benefit - What a noble goal! I had no idea. ^^
I am extracting DNA of a weevil and I get as shmear in my gel too. HMM I will look into getting fresher samples
Your as interesting brain wise as I am,,,, what about a crisper? Amino acids to proteins I think is 16 or 17 Amino acids,, your good,, I'm rusty practice on this stuff because I owned a plumbing heating business locally that would get me home by 6pm for dinner,, glad to study with you..
Oooh, this is exactly what we did for our Biochemistry lab! :D Well, with E.Coli anyway. no 27 steps needed.
Would it be possible to lyophilize frozen spiders prior to extraction? I work with semi-recalcitrant plant tissues (conifer needles and twigs) and I've found that it makes specimens wonderfully brittle, allowing for more thorough homogenization.
Sure, frozen and lyophilized spiders would be an absolute dream, but getting that is pretty hard. Most of the people crazy enough to raise widows don't have a freeze dryer around. But if that was an option I'd happily take it
your nanodrop readout at 9:28 has a horrible 260/280 value. you'd expect this ratio to be 1,8-2 and 0,56 already tells you that there is wayyy more protein remaining than normal. not saying that i wouldnt have looked past that red flag and still used the DNA in hopes of a successful PCR though. also, i dont think you necessarily need one of these kits to isolate the DNA. ive had success extracting DNA from eucaryotic cells by simply boiling them for 30 mins with some proteinase K thrown on top and you can isolate DNA from mouse tail tips for genotyping with a very very simple protocol, no kit involved. maybe you could try a simple protocol such as this www.ncbi.nlm.nih.gov/pubmed/20204529 (bypass evil springer with sci-hub) and try adding different proteinase K concentrations to the buffer since you already have it. dont forget to kill it afterwards though, you dont want to throw your polymerase in there. also, maybe try freezing the spider in lN2 and crash it with a hammer - doesnt get any more fine than that.
I came as fast as I could
Will their be anymore videos on this subject since I can't find any way of putting them in proper order and never got to see if it ever worked
Yup I'm still working on it. I expressly said at the start of the project that it's really hard and slow and updates will take time. I've been working diligently in the background but have arrived at the conclusion that trying to do this by hand is stupid. SO I'm working on redesigning everything so I can just send it to a synthesis company, have them make the complete full plasmid, and then send it to me ready to be put directly into yeast. But since it's such a repetitive structure, it's taking time to design something that's synthesizable. Also, I need to eat which means I need to make videos. And this is too slow and too boring to watch me fail over and over, so I've been working on other things and making other videos to pay for the cost of just having it made.
Dude, it's just so cool!
can someone explain to me why exactly you would want to make spider silk?
its as strong or stronger than kevlar and when this is done I'll be able to grow it continuously using basically only sugar water as the starting material. But unlike kevlar I can modify it in 1000 ways to have whatever other properties I want.
holy shit what have i been doing all my life
So I want to extract dna from my baby brother should I cut off a toe and make a paste from that or grind up the whole thing
Also this is a joke I don’t actually want extract any dna. I’m just really hungry.
If I put the CBD/THC genes into yeast and brew beer with it, or bake something, would I get high?
Also, would these fungi be illegal in a country where cannabis is illegal?
Good luck with that. My friend runs a company that does that and it took them years to get it working and 10 mil investment. But yes it's probably get you high
Is there a way that I can get spider DNA other than by hand
Hi, In this method at what rmp do you spin the columns?
If i were to do this, id use a species i could raise in captivity, and use captive bred individuals for all manor of research. Behaviors, anatomy, genetics, ect. By genetics i mean stuff like this, but also heritaty and what not. Possibly other kinda of research into them but thats besides the original point, which was that id use a captive population as to not withdraw from the wild stock and have more control over the dna i use.
@@XMysticHerox im aware, fruit flies was actually my go to as i understand their biology a bit better than other model species.
Does nobody else see a couple tubes move on their own at 5:05?
I require updates on this.
Warm dark places tend to have arachnids.
Is this your main job?
Very cool!
All of your videos are great, and this one is no different... But spiders and cake in the same video, your killing me man. What did cake ever do to you.
Guess who's back....back again
How are you going to isolate the silk protein gene from the spider?
Check out the PCR video. I explain that in detail.
for animal cells, sonication is far more efficient
For What you will use this dna?