Is it possible to make glowing plants at home with basic lab eq? (PCR machine, Centrefuge, Incubator)

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Anonymous Sun Apr 26 20:51:34 2015 No.7221096 [Reply] [Original]

Is it possible to make glowing plants at home with basic lab eq? (PCR machine, Centrefuge, Incubator)

>>	Anonymous Sun Apr 26 20:54:13 2015 No.7221104 >>7221096 Why is Kurisu so fucking autistic? Can't she just wear her jacket like a normal fucking person?

>>	Anonymous Sun Apr 26 20:57:54 2015 No.7221111 >>7221104 Niggah I dont care about her. I just wanna know if I can make some plants. Im sick of fucking around with e.coli.

Anonymous Sun Apr 26 21:02:13 2015 No.7221118

>>7221111
But there's no fucking reason to wear it like that. If doesn't cover your shoulders or chest which is the main areas of heat transfer so it's just an unnecessarily limiting movement and causing annoyance around her arms. She might as well just take it off completely.

>>	Anonymous Sun Apr 26 21:05:52 2015 No.7221125 >>7221111 Yes. Lux cassette. 35S strong promoter. Agrobacterium tumefasciens. Good to go.

>>	Prof Sai Sun Apr 26 21:09:30 2015 No.7221129 >>7221104 >Why is Kurisu so fucking autistic? Can't she just wear her jacket like a normal fucking person? Women + clothes. This is the only strange thing you have ever noticed?

>>	Anonymous Sun Apr 26 21:23:11 2015 No.7221146 >>7221125 >Agrobacterium tumefasciens. Wait, so its not the plants that are going to glow, its the pathogen I infect them with? Try again

>>	Anonymous Sun Apr 26 21:36:56 2015 No.7221161 >>7221096 if you have to ask, you cant do it.

>>	Anonymous Sun Apr 26 21:46:40 2015 No.7221170 Impossible

>>	Anonymous Sun Apr 26 22:20:12 2015 No.7221205 >>7221146 The Agrobacterium houses and transfers your DNA to the plant host

>>	Anonymous Sun Apr 26 23:12:19 2015 No.7221281 >>7221205 Alright, one more question.. Is there a way I can get the 35S primer so I can isolate the promotor from gmo? Or better yet, where can I get the promotor?

>>	Anonymous Mon Apr 27 00:16:20 2015 No.7221361 >>7221281 https://www.addgene.org/12588/ You're going to need puromycin as well for the agrobacterium transformation.

>>	Anonymous Mon Apr 27 01:07:55 2015 No.7221439 >>7221361 >416$ Guess I'll go back to my E.Coli transformations.

>>	Anonymous Mon Apr 27 01:16:41 2015 No.7221450 >>7221104 >>7221118 maybe her butt hurts??

>>	Anonymous Mon Apr 27 02:01:32 2015 No.7221503 >>7221361 >puromycin not op but what is this for?

>>	Anonymous Mon Apr 27 02:12:51 2015 No.7221512 >>7221503 The vector with your luminescence gene also has a gene for antibiotic (puromycin) resistance. After transformation you apply your antibiotic to kill off the untransformed bacteria.

Anonymous Mon Apr 27 08:22:13 2015 No.7221876

>>7221439
Where are you getting the $416 from? I'm seeing $65. Also, I don't know if you've looked, but thermal cyclers, centrifuges, and incubators aren't exactly cheap. Shipping and handling are bound to be expensive to.

Barrier of entry costs to at home biohacking is absolutely obnoxious. Then you have to deal with sterility issues. You should look to see if there's a DIY bio community in your area. They'd probably the infrastructure you need and the expertise to help you figure out what you want to do.

>>	Anonymous Mon Apr 27 08:59:08 2015 No.7221909 >>7221118 >>7221104 >accusing an imaginary object of autism >being absolutely autistic about it

>>	Anonymous Mon Apr 27 09:27:16 2015 No.7221949 >>7221118 >>7221909 rekt

Anonymous Mon Apr 27 13:29:08 2015 No.7222226

>>7221512
Also not OP and only highschoolfag here.
Does the plant have to be young when it's infected to allow the infected cells to multiply? or will there just be enough bacteria to infect a shit ton of the cells in a fully grown plant? When the cells divide will they transfer the luminescence gene with it?

>>	Anonymous Mon Apr 27 13:44:47 2015 No.7222257 >>7221512 also, can I safely turn my rat into a glow-in-the-dark pet

Anonymous Mon Apr 27 13:46:34 2015 No.7222261

>>7222226

Typically what's done is you regenerate the plant from one of your transformed cells. So yeah, pretty damn young.

Transforming a plant cell involved inserting your gene of interest into the plant's genome. Once that happens, DNA replication and cell division will take your gene along with it.

>>	Anonymous Mon Apr 27 13:48:37 2015 No.7222265 >>7222257 http://agapakis.com/hssp/images/GFP_animals.png https://en.wikipedia.org/wiki/Green_fluorescent_protein

>>	Anonymous Mon Apr 27 13:53:22 2015 No.7222272 >>7222261 >>7222265 I love you. You've literally just given me more inspiration than any teacher I've ever had. Thanks to you I'll get shit done during my undergrad and maybe start a club for this kind of stuff.

>>	Anonymous Mon Apr 27 14:10:40 2015 No.7222301 >>7222272 Clubs are great. I'd recommend joining one or two to anyone. I'd credit 90% of my social skills to my college clubs.

Anonymous Mon Apr 27 14:26:08 2015 No.7222319

>>7222226
depends on the plant

if you're doing it in Arabidopsis, you use young flowering plants with lots of clusters of buds (you let it grow and flower a week or so, then chop off the primary apex to encourage branching), then you dip the aboveground plant in an Agro suspension. search for "floral dip arabidopsis" to get protocols, it's dirt simple

if you're doing it in other plants you probably have to generate embryonic or root callus, which is a pain in the fucking ass, and sterility is a huge problem. i'm doing it right now in rice, the protocol itself is dirt simple, it just takes F O R E V E R. selection of transformed rice callus takes like ten weeks on its own, and that's before you've even regenerated the plant.

honestly i'd just do it in Arabidopsis, it's way easier than fucking around with callus. if there's any plant biology labs at a college in the area, bribe a grad student with some beer to get materials like agro and plasmids and wildtype seeds, it's way cheaper than ordering it.

>>	Anonymous Mon Apr 27 14:27:57 2015 No.7222323 >>7221118 steins;gate takes place in the summer, conserving heat is the last thing she'd want to do

>>	Anonymous Mon Apr 27 14:35:15 2015 No.7222336 >>7222319 So from what I understand the plasmid can be just added to a culture of agrobacterium, and by incubating it the bacteria will transform? excuse my lack of knowledge but doesn't the plasmid come already inside bacterial cells?

Anonymous Mon Apr 27 14:46:38 2015 No.7222360

>>7222336
"plasmid" is a term for a piece of circular DNA. plasmids in general are found naturally in bacteria. plasmids can also be constructed in the lab artificially and introduced into bacteria.

in order to transform into a plant, you need to generate a plasmid capable of being transformed into the plant. this involves a couple enzymes used by Agrobacterium in the process of transformation as well as the actual DNA to be transformed, which is flanked by two pre-specified regions.

you transform your DNA of interest into the plasmid (typically you just already have the plasmid or you get it from someone like addgene or another lab) and introduce it to Agrobacterium cells which have been made competent for the uptake of DNA (a pain in the ass, and requires an electroporator machine). then you select the Agro for transformed cells using antibiotic-laced growth media.

you grow the transformed Agro up in a culture and dip the flowers in the solution (generally, there's more steps but not relevant here, the protocol is available online). the agrobacterium is capable of inserting foreign DNA into the cells of plant species and facilitating incorporation into the cell's genome.

>>	Anonymous Mon Apr 27 14:49:52 2015 No.7222366 >>7222360 Thank you so much. so the plasmids just come in a pure form of DNA? i don't have to extract them from bacteria?

Anonymous Mon Apr 27 14:54:47 2015 No.7222374

>>7222366
i left out a lot of steps

so like, if you're lucky you'll get an aqueous solution of the base plasmid, either from addgene or from another lab. you need to insert your DNA of interest into that base plasmid (lots of methods, you can do old school ligations, gateway transformations, and newer methods like SLIC/Gibson) and then transform it into E. coli. You grow up lots of E. coli and then extract the plasmid from the bacteria. if you're not lucky, you'll get the plasmid in the form of E. coli cells (which contain the plasmid) struck out on a plate, and you need to grow up a culture of them and extract the plasmid.

Then, you take the extracted plasmid and transform into Agrobacterium.

Anonymous Mon Apr 27 14:56:53 2015 No.7222379

>>7222366
take a look at these protocols:
http://www.plantpath.wisc.edu/fac/afb/protocol.html
http://openwetware.org/wiki/Miniprep
http://openwetware.org/wiki/Bacterial_transformation

keep in mind that Agrobacterium has to be done by electroporation, you can't do chemical competence with Agro like you do with E. coli

>>	Anonymous Mon Apr 27 14:57:54 2015 No.7222381 >>7222379 >Agrobacterium transformation fixed

Anonymous Mon Apr 27 15:14:46 2015 No.7222402

>>7222366
If you're inexperienced with basic molecular techniques and trying to do this at home, you're going to be dropping a shit ton of money on equipment and reagents to attempt a tedious protocols you might not even enjoy performing and will undoubtedly fuck up many times.

I'd recommend volunteering in a lab that already does this type of work. You get the training without sinking your personal finances while getting valuable documented experience that you can place on your CV.

>>	Anonymous Mon Apr 27 15:26:44 2015 No.7222416 >>7222402 I found it! http://www.bio-rad.com/en-ca/sku/166-0405edu-pglo-plasmid let's make some glow-in-the-dark shit /sci/!

>>	Anonymous Mon Apr 27 15:28:37 2015 No.7222419 I want to buy property in a county where GMOs are banned and plant some glowing arabidopsis in my yard. Fucking haters.

>>	Anonymous Mon Apr 27 15:29:51 2015 No.7222423 >>7222402 >>7222416 http://www.bio-rad.com/en-ca/product/pglo-bacterial-transformation-kit and this one's only 150 bucks!

>>	Anonymous Mon Apr 27 15:36:55 2015 No.7222437 >>7222419 Sounds like a great way to introduce a transgenic invasive weed into the environment.

Anonymous Mon Apr 27 16:15:53 2015 No.7222524

>>7222437
eh, it'll probably die out in a couple generations

most people stick GFP in under a hihgly active constitutive promoter, which will represent a non-negligible metabolic load for the cell. outside the lab, there will be a selective force against maintenance of the transgene.

>>	Anonymous Mon Apr 27 16:27:34 2015 No.7222558 >>7222524 True. But that's assuming that there are wild type Arabidopsis present in the ecosystem to compete against. If it's novel with no niche competitors, it's pretty much there to stay. Ain't no putting that toothpaste back in the tube.

>>	Anonymous Mon Apr 27 16:40:52 2015 No.7222589 >>7222558 there's already Arabidopsis in Europe and North America, it's a pretty ubiquitous weed

>>	Anonymous Mon Apr 27 17:14:03 2015 No.7222645 >>7221096 I read this as 'glowing pants'.

>>	Anonymous Mon Apr 27 21:17:46 2015 No.7223125 >>7222374 >>7222379 So it seems easy enough to use agrobacterium to transfer genes into plants. how do they get the plasmids into mice?

Anonymous Mon Apr 27 23:44:09 2015 No.7223349

>>7223125
They use different strategies for eukaryotes.
Microinjection of transposons, electroporation, CRISPR/Cas9, viral transfections.

http://www.cell.com/abstract/S0092-8674%2814%2901163-5

http://www.ncbi.nlm.nih.gov/pubmed/24625778

http://www.ncbi.nlm.nih.gov/pubmed/7657292

>>	Anonymous Mon Apr 27 23:49:57 2015 No.7223363 >>7223349 For clarification. When I say eukaryotes, I meant in comparison to the Agrobacterium transformation.

>>	Anonymous Mon Apr 27 23:52:33 2015 No.7223367 >>7222272 In my highschool AP Bio class, we actually did this with e coli.

>>	Anonymous Mon Apr 27 23:53:24 2015 No.7223370 how do i infect myself to glow in the dark

>>	Anonymous Mon Apr 27 23:53:49 2015 No.7223372 >>7223125 That's WAY harder. Either you try to get it into a virus or you use these CRISPR/Cas9/TALENS thing that's fairly new and I don't really understand.

>>	Anonymous Tue Apr 28 00:03:00 2015 No.7223385 >>7222374 How to I ensure the Agrobacterium has transformed correctly. Doesnt the lux gene have to be in a special spot to ensure insertion into the plant?

Anonymous Tue Apr 28 00:22:45 2015 No.7223434

>>7223385
That's what the antibiotic resistance gene on the plasmid is for. If it survives the selection process, then it is assumed to be successfully transformed. This is often confirmed via plasmid extraction and restriction digest.

The lux gene is downstream of the promoter housed on the plasmid, which is its own separate structure from the bacterial genome. Agrobacterium T4 DNA ligases mediate the plasmid insertion into the host genome.

>>	Anonymous Tue Apr 28 00:24:45 2015 No.7223437 >>7223434 Ah, coolio. I wasnt sure if I needed to do somthing special, but it just injects the plasmid? So once sucsessful transformation occors it doesnt need anything else?

Anonymous Tue Apr 28 00:36:13 2015 No.7223448

>>7223437
You're basically coopting its existing DNA injection mechanism, the type IV secretion system.

Even if you get the DNA inside the host, shit can still hit the fan. You still have do some sort of transformation verification of the plant itself, DNA extraction and digest, and if you're in a formal lab setting, also verify the expression of the protein of interest.

The transformation rate of the plant itself can be stupid low. I worked in a plant lab for a year or two. I got really frustrated with it. Longer generation times than microbes and they're complex fucking systems.

>>	Anonymous Tue Apr 28 01:31:12 2015 No.7223518 >>7223448 dont listen to this fuck u cant use any old plasmid, it needs to be the tiplasmid

>>	Anonymous Tue Apr 28 01:36:22 2015 No.7223526 Niggah just let your plants absorb water with a fluorescent agent. Unless you actually want to GMO them.

>>	Anonymous Tue Apr 28 02:53:15 2015 No.7223587 >>7223518 Weird. It's almost as if the type IV secretion system is encoded and regulated by the Vir genes located on the Ti plasmid.

>>	Anonymous Tue Apr 28 13:19:22 2015 No.7224154 >>7223587 Where 2 get ti plasmid

>>	Anonymous Tue Apr 28 15:07:37 2015 No.7224342 File: 18 KB, 400x321, 1429108284351.gif [View same] [iqdb] [saucenao] [google] >>7224154 from the plasmid library

>>	Anonymous Tue Apr 28 15:46:23 2015 No.7224404 >>7224342 can someone find me a Ti plasmid with pGLO or some GFP gene in it?

>>	Anonymous Tue Apr 28 15:49:53 2015 No.7224412 >>7224404 Nigger, its simple. Isolate the plasmid from the agrobacteria and cut it with a restriction enzyme and put the pGLO in yourself. Just make sure you find the right RE. Thats where I'm stuck at.

>>	Anonymous Tue Apr 28 15:52:19 2015 No.7224420 >>7224404 My lab has eGFP in agro already if you want me to sneak a little out for ya

>>	Anonymous Tue Apr 28 15:56:24 2015 No.7224431 >>7224420 You'd be the hero of the year, but I wanna do this the hard way. also international shipping's a bitch

>>	Anonymous Tue Apr 28 15:57:05 2015 No.7224433 >>7224420 I wonder what USPS thinks about shipping bacteria...

>>	Anonymous Tue Apr 28 15:57:23 2015 No.7224435 >>7224431 What country do you live in?

>>	Anonymous Tue Apr 28 15:59:01 2015 No.7224439 >>7224433 I'll stick it in a sock so that they don't know what it is

>>	Anonymous Tue Apr 28 16:02:29 2015 No.7224446 >>7224435 Canada. Also the problem is once i get it, i've only got the lifespan of the bacteria to find a place that'll let me use their equipment T_T

>>	Anonymous Tue Apr 28 16:04:51 2015 No.7224450 >>7224446 All you have to do is make a few plates for the bacteria to survive on (beef and yeast extract, sucrose) and transfer them once a month. Make 6 plates (probably like 15mls each) and you can keep the agro for half a year

>>	Anonymous Tue Apr 28 16:13:53 2015 No.7224459 >>7224439 I would if I were just trying to whip something off for the sake of having a cool houseplant. I wanna struggle through this the hard way so I can have something I made to show off before I finish high school and it stops being impressive.

>>	Anonymous Tue Apr 28 16:16:05 2015 No.7224463 >>7224439 But I'll gladly trade contact info with you in case i change my mind!

Anonymous Tue Apr 28 16:30:25 2015 No.7224494

>>7223125
you extract cells from embryos, modify the cells, inject them into other extracted embryos, implant the embryo in a mouse, do all that a bunch of times and get lots of chimeric mice.

you cross your fingers and hope one of the chimeric mice had the altered cell give rise to the germline cells, in which case you mate that mouse to a wildtime to get a segregating line and then select for homozygosity. if you don't get a chimera with a germline mutation you just keep trying until you do.

>>	Anonymous Tue Apr 28 16:33:22 2015 No.7224500 >>7223448 that's why you only join plant labs that study phenotypes visible in seedlings root assays are a fucking blessing, i feel really sad for the people i know who study meiosis phenotypes in late senescence.

>>	Anonymous Tue Apr 28 16:34:23 2015 No.7224503 >>7224500 also it could be worse. some people use trees as their model organism.

>>	Anonymous Tue Apr 28 16:44:45 2015 No.7224516 File: 25 KB, 343x380, General Recoome thinks that is pretty neat.jpg [View same] [iqdb] [saucenao] [google] >/sci/ having an actual biotechnology, molecular biology, and genetics thread >no shit posting at all I never thought I would see the day.

>>	Anonymous Tue Apr 28 16:51:47 2015 No.7224529 >>7224420 OP here, if you're serious I will LOVE you. I got some of dem bitcoins too. reptillianlizzard@gmail.com

>>	Anonymous Tue Apr 28 16:53:15 2015 No.7224532 >>7224529 I'm not exactly sure how I would ship it though

>>	Anonymous Tue Apr 28 16:54:14 2015 No.7224537 >>7224404 You know what I want? A Ti Plasmid with the T-DNA broken and a restriction site right where my lux genes need to be inserted. Does this exist?

>>	Anonymous Tue Apr 28 16:59:50 2015 No.7224547 >>7224537 Im not sure, but it would sure make it easy. Im looking on addgene for it.

Anonymous Tue Apr 28 17:01:35 2015 No.7224550
File: 1.19 MB, 1520x2688, 2014-09-25 17.12.59.jpg [View same] [iqdb] [saucenao] [google]

>>7224537
you actually probably want gfp like anon offered, not luciferase - luciferase needs a substrate, but gfp only needs uv light. i mean, luciferin is dirt cheap, but it's still one more thing to buy/acquire

>>7224516
we bio anons are always around, it's just /sci/ never talks about biology so all we do (or at least all I do) is shitpost in the math and physics threads

also, for shits and giggles here's a picture of a 35S::mCherry Arabidopsis seedling I took a couple months ago

Anonymous Tue Apr 28 17:05:40 2015 No.7224554

>>7224446
one time i stuck a plate of e coli in the fridge and came back to it a couple months later and still managed to get a couple viable cells off of it. don't know if you can do the same thing with agro, but it's worth a shot.

plus, if you get your hands on some glycerol, you can just make frozen stocks. those fuckers will last for a while.

>>	Anonymous Tue Apr 28 17:08:08 2015 No.7224559 >>7224529 I sent you an email >>7224554 Assuming he has a neg 80

>>	Anonymous Tue Apr 28 17:08:22 2015 No.7224560 >>7224554 How long can an e.coli plate last at -20C?

>>	Anonymous Tue Apr 28 17:13:27 2015 No.7224570 >>7224559 you can store glycerol stocks at -20, it's just that they only last a couple months rather than indefinitely >>7224560 don't freeze the plate, that'll just kill the cells.

>>	Anonymous Tue Apr 28 17:17:42 2015 No.7224572 Thanks /sci/, now i just gotta buy a cheap micro centrifuge, some bacteria and a freezer, and borrow some reagents from school, and I'll have a really interesting hobby for a first-year undergrad!

>>	Anonymous Tue Apr 28 17:28:22 2015 No.7224582 >>7224420 Not with the intention of creating a shitstorm of people begging you for bacteria... but... plsplsplspls thomassoutar2@gmail.com

>>	Anonymous Tue Apr 28 17:29:34 2015 No.7224584 >>7224420 what's your selection system? is it antibiotic or one of those nifty metabolic systems? it might be hard for anons to get their hands on antibiotic stocks

>>	Anonymous Tue Apr 28 17:36:56 2015 No.7224594 http://www.ebay.ca/itm/Microcentrifuge-Scllogex-D1008-mini-centrifuge-5000RPM-economic-free-shipping-/331438227001?pt=LH_DefaultDomain_0&hash=item4d2b41fe39 Is this one any good? will it work with normal sized centrifuge tubes?

>>	Anonymous Tue Apr 28 17:38:15 2015 No.7224596 >>7224584 We use antibiotics as selection agents. Off the top of my head, the selective agents for the eGFP is kan pen >>7224582 I'm assuming you want the same thing? Email me what you want and what state you live in and I'll email you back

>>	Anonymous Tue Apr 28 17:39:17 2015 No.7224600 >>7224596 >forgetting the email address fug thegoyknowshutitdown@gmail.com

>>	Anonymous Tue Apr 28 17:39:35 2015 No.7224601 >>7224596 I don't have your e-mail

Anonymous Tue Apr 28 17:40:43 2015 No.7224604

>>7224594
that's a little underpowered. you'll have a really hard time pelleting cells, those are more for getting all the moisture down to the bottom of the tube if you drop it.

http://www.ebay.ca/itm/Eppendorf-5415-Microcentrifuge-centrifuge-laboratory-/190522029385?pt=LH_DefaultDomain_0&hash=item2c5bff7549 this is more along hte lines of what you need.

>>	Anonymous Tue Apr 28 17:42:58 2015 No.7224607 >>7221104 >>7221111 >>7221118 >2015 >watching Japanese cartoons ishiggy diggy

>>	Anonymous Tue Apr 28 18:31:40 2015 No.7224655 >>7224572 If you have access to 3d printing you can make a centrifuge out of a dremel tool and a specially printed part. Google dremelfuge.

>>	Anonymous Tue Apr 28 18:40:07 2015 No.7224668 >>7224655 Thank you so much, these at-home remedies are perfect for a poor high school student. I've also heard you can incubate things in your arm-pit if you're willing to hold that there long enough.

>>	Anonymous Tue Apr 28 18:55:18 2015 No.7224687 Does anyone know how to isolate the TiPlasmid and change the tuna region

>>	Anonymous Tue Apr 28 18:56:55 2015 No.7224689 >>7224668 Well you need to incubate them for hours upon hours, but if you're willing...

>>	Anonymous Tue Apr 28 18:59:02 2015 No.7224694 >>7221104 because anime

>>	Anonymous Tue Apr 28 19:03:39 2015 No.7224698 >>7224687 >>7224687 >mfw autocorrect I meant TDNA

>>	Anonymous Tue Apr 28 19:13:43 2015 No.7224709 >>7224689 Will a desk lamp and a thermometer work?

>>	Anonymous Tue Apr 28 19:48:32 2015 No.7224754 >>7224550 If you use the bacterial lux cassette, the substrate already produced within the same operon.

Anonymous Tue Apr 28 19:54:30 2015 No.7224768

>>7224709
Agrobacterium is often grown at 22 C. Stuff like E. coli is incubated at 37 C. Check what temperature your bug likes best. Depending on what you've got, a hot water bath might be better for incubation. If you can set up some sort way for your flask to be agitated during the incubation process, that would be even better.

>>	Anonymous Tue Apr 28 19:57:27 2015 No.7224776 >>7224550 Damn son, purty ass transformation. Good on you.

Anonymous Tue Apr 28 19:57:33 2015 No.7224777

>>7224668
>>7224709
take a gander at this: http://www.seriouseats.com/2010/04/cook-your-meat-in-a-beer-cooler-the-worlds-best-sous-vide-hack.html

also 37c will kill agro right quick. and remember that it takes an extra day to grow, it's not an overnighter like e. coli

>>7224687
minipreps to isolate and purify, then any of various methods for cloning. see>>7222374

>>	Anonymous Tue Apr 28 19:58:33 2015 No.7224778 >>7224776 (it wasn't mine, it was a reporter line we got from someone else. i wish it was mine)

>>	Anonymous Tue Apr 28 19:59:56 2015 No.7224781 >>7224754 oh huh, i didn't know. nevermind, ignore me.

Anonymous Tue Apr 28 20:08:46 2015 No.7224792

>>7224777
Shit. That's fucking clever. I wonder how long the system would be able to retain that heat. If you left it outside in the sun, I bet it would stay pretty steady.

>>7224781
I used to do microinjections with the luc. It was nice because it so much smaller than lux but addition of the substrate was always a pain in the ass for long term imaging.
How would you add it for the plant imaging?

>>	Anonymous Tue Apr 28 20:50:36 2015 No.7224864 >>7224777 my lab is going to be the most ghetto thing. >centrifuge out of a dremel tool and an empty paint bucket for safety >incubator made out of a beer cooler >no glassware yet, i'll have to get creative for that and wellplates

>>	Anonymous Tue Apr 28 21:16:04 2015 No.7224893 >>7224864 I have friends who have used an aquarium turned on its side as a trailer-trash biosafety cabinet for mushroom spore inoculation. The toughest part about working at home is keeping your stuff asceptic.

Anonymous Tue Apr 28 21:16:15 2015 No.7224895

>>7224792
from what i've heard if you insulate the beer cooler well (drilling holes in the lid and filling the interior of the lid with expanding foam, like that stuffit stuff), you can hold your heat loss to about a degree C an hour.

also most of the applications of luciferase i know of in plants involve supplying it in the water and waiting for the plant to take it up through the roots. see: https://youtu.be/d3oejKRgiOM?t=2237

>>	Anonymous Tue Apr 28 21:21:18 2015 No.7224900 >>7224895 (also no, before anyone asks, you can't do that kind of imaging in your home, you need a fifty thousand dollar cooled CCD camera taking five minute exposures to get those images)

>>	Anonymous Tue Apr 28 21:26:09 2015 No.7224908 >>7224893 This. You'll really need a sterile environment to do a transformation to prevent contamination. Also, depending on what species everyone is trying to work with, your rate of transformation may be 1/1000

Anonymous Tue Apr 28 21:31:25 2015 No.7224911

>>7224908
eh, i do transformations on a very unsterile bench all the time, no flame needed

as long as you have sterile media and sterile tips/tubes, you almost never have to worry about your bench being sterile

do you mean transformation rate of the plants or the bacteria?

>>	Anonymous Tue Apr 28 21:34:40 2015 No.7224915 >>7224777 Is there a restriction site on the Ti Plasmid in the t-DNA? Thats where I am stuck

Anonymous Tue Apr 28 21:37:15 2015 No.7224919

>>7224915
generally, you can't know without looking at the plasmid map.

specifically, the answer is almost certainly yes - restriction digests are the bread and butter of cloning. even if you're doing some horribly complicated cloning scheme using proprietary enzyme mixes and whatnot, you're going to start with a restriction digest.

Anonymous Tue Apr 28 21:41:33 2015 No.7224924

>>7224919
Alright. Thanks for the help. When I made this thread I was expecting a bunch of shit posting, but I learned so much

Heres my plan

1.) Aquire 35S and Lux cassette from add gene
2.) Somehow isolate the individual lux cassette genes and get promoters in between them
3.) Isolate Ti Plasmid
4.) Find a restriction map... Somwhere of the Ti Plasmid, cut in the middle of the T-DNA region and insert my Primer and Lux cassette
5.) Hope it works

I have a more detailed write up, but this is the general idea that I am thinking of.

Anonymous Tue Apr 28 21:50:41 2015 No.7224933

>>7224924
here's a couple really important questions for you to think about. you say you have a more detailed writeup and i believe you do, but i just want to make sure you've thought about these things. you dont necessarily ahve to answer these questions in the thread, i just want to make sure you're thinking about them.

how are you going to amplify and isolate the DNA for the promoter and gene you want to insert into the Ti plasmid? there's a couple ways you could do it, each with their own benefits and drawbacks

how are you going to insert those genes into the Ti plasmid in a manner that ensures they are one whole, stable unit? is it an enzymatic reaction? if so, where are you going to get the enzymes and buffers? if not, how are you going to do the reaction?

how are you going to insert the completed plasmid into bacteria, either E. coli or agrobacterium?

i strongly recommend browsing through a lot of the transformation and cloning protocols on openwetware so that you're familiar with the techniques and steps you'll need to go through

Anonymous Tue Apr 28 21:57:57 2015 No.7224942

>>7224933
1.) Amplification will be done with PCR. Im still not sure how I am going to divide the lux genes. I'll email my bio prof.
2.) This is part of what I am trying to solve. From what I hear the plasmid is too big for just a digest.
3.)Electroc competence via the openwetware protocol.

Half of the fun is putting together the puzzle. I'd say I'm half way there. Still have some major holes.

Heres my more complete write up:http://pastebin.com/45W8fB2S

>>	Anonymous Tue Apr 28 21:58:42 2015 No.7224945 File: 8 KB, 222x216, fad.jpg [View same] [iqdb] [saucenao] [google] What incubator do u fuks think I should buy. Used off eBay ofc

>>	Anonymous Tue Apr 28 22:14:24 2015 No.7224959 >>7224942 nigga if you get this working you've gotta sauce me some of those germs! I don't have the equipment to do this stuff D':

Anonymous Tue Apr 28 22:55:56 2015 No.7225030

>>7224942
Right on bruv. Just looked at your protocol. I'm not sure if puromycin will act as selective agent for Agro. Someone else may have to confirm.

This protocol uses streptomycin or kanamycin, which may be cheaper.

http://www.bio.utk.edu/cellbiol/prot/Agrobacterium-Transformation.pdf

>>	Anonymous Tue Apr 28 23:08:42 2015 No.7225050 >>7225030 the main issue that everyone seems to be forgetting is that the tiplasmid is huge and restriction enzymes and ligase arent going to cut it.

>>	Anonymous Tue Apr 28 23:16:48 2015 No.7225058 >>7225050 the native Ti plasmid is too big, yes. that's why labs use plasmids that have cut down the Ti plasmid to its minimal elements for functionality and transform into Agro containing the whole, functional plasmid

>>	Anonymous Tue Apr 28 23:25:56 2015 No.7225065 >>7222360 >a pain in the ass, and requires an electroporator machine Can't do heat uptake with agrobacterium?

>>	Anonymous Tue Apr 28 23:52:48 2015 No.7225093 >>7225058 nobody has told op where too get the lab plasmid doesnt matter though cause that faggot doesnt know what they are doing.

>>	Anonymous Wed Apr 29 00:00:45 2015 No.7225101 >>7225093 See >>7224420. Thanks for all of the valuable insight, champ. >>7225065 I've seen protocols where the heat shock is used. I would assume that the electroporation efficiency would be higher though.

>>	Anonymous Wed Apr 29 00:04:03 2015 No.7225104 >>7225101 nigger do u really think annon is going to give op free shit? how about u find a good internet source to buy it at

Anonymous Wed Apr 29 00:14:10 2015 No.7225111

>>7225104
It's close to $400 to buy that shit from a supplier. OP wanted help for diybio. Help from the community was given. Why are you surprised?

Maybe if you weren't such an insufferable bitch, people around you would be willing to help you out more too.

Anonymous Wed Apr 29 00:18:07 2015 No.7225114

>>7225104
OP here. You're right, I dont know anything, but I dont think you do either, so shut the fuck up :^)

I like how you decided for me and the other annon if we are going to exchange plasmids for us.

I was in email correspondence with him, and I'm pretty sure he will help out. I haven't gotten a reply but I can always hope.

Please get checked for autism.

>>	Anonymous Wed Apr 29 00:40:40 2015 No.7225132 >>7225111 Hey there, On the off chance annon doesn't help me, do you have any idea for shrinking the plasmid or getting somthing viable for the plant agro transformation?

Anonymous Wed Apr 29 02:43:20 2015 No.7225229

>>7224911
Transformation of the plants themselves. The way I do them is I take cuttings of the plant, co-cultivate it with the agro in an incubator for 2-3 days, then move it to callus inducing media for a few weeks, then move it to shoot inducing media. Of course with seeds it's much easier

>>	Anonymous Wed Apr 29 03:28:55 2015 No.7225263 File: 13 KB, 188x268, download (3).jpg [View same] [iqdb] [saucenao] [google] >>7221104 >>7221096 >chinese cartoons >realistic

>>	Anonymous Wed Apr 29 08:36:24 2015 No.7225507 >>7225229 oh ok, i see what you mean. what plants do you work with?

>>	Anonymous Wed Apr 29 12:10:07 2015 No.7225749 >>7225132 For eventual cost and labor purposes, I'd recommend going through addgene and selecting established plasmids for transformation. Look into the pGREEN line.

>>	Anonymous Wed Apr 29 12:20:41 2015 No.7225776 >>7225263 cheap bait

Anonymous Wed Apr 29 12:20:51 2015 No.7225778
File: 35 KB, 338x501, mrna-fig-3.gif [View same] [iqdb] [saucenao] [google]

>>7224942
How are you going to purify the digested fragment from your restriction digest? Have you looked into how to do a gel extraction and what materials you'd need?

How are you going to insert digested gene into your plasmid? Are you going to use a ligase? If you are, remember that ligase buffer is super sensitive, the ligation reaction needs free ATP to work and that degrades fast if you don't handle hte buffer carefully.

i recommend drawing a diagram of your whole cloning scheme. pic related is a really quick and dirty drawing o fa cloning scheme - the idea is to clearly identify all the different plasmids you're using as sources for DNA, the manipulations you're performing, how the different pieces are being combined, and what hte final produt will look like.

>>	Anonymous Wed Apr 29 12:26:30 2015 No.7225797 >>7225507 I work with a plant called sticky nightshade. Its a pain in the ass to work with because of all the spines it has on it

>>	Anonymous Thu Apr 30 01:30:31 2015 No.7227054 File: 2.00 MB, 3264x2448, IMG_0029.jpg [View same] [iqdb] [saucenao] [google] >>7221096 >wanting to make glowing plants >asking how >thinking you can do it if you have to ask. OP, are you retarded?

>>	Anonymous Thu Apr 30 09:59:42 2015 No.7227524 >>7227054 it's actually not that hard if you have all the materials and you're willing to look up the protocols i could teach a high schooler how to do it

>>	Anonymous Thu Apr 30 19:19:12 2015 No.7228567 >>7227524 no op is retaded cause he wasn't willing to look it up. probably goes to a cc and thinks he's hot shit.

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/sci/ - Science & Math