Tissue Culture/DNA transformations

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Loki7

Well-Known Member
After seeing what you guys think about LEDs, I don't know if anyone would be interested in this, but here goes...

I've started doing tissue cultures for micro propagation and I had some really good results. So I did some research in the advances in DNA manipulation and was surprised at how far things have come.

See the attached pics. One is the manufacture 'cheat sheet' for preparing DNA samples and the other is the directions for my vaporizer. The vaporizer has 3 more steps than the DNA prep! (okay, it is harder than smoking a vaporizer, but not too much)

I've used this (http://www.jbc.org/cgi/content/full/279/38/39767) as a guide, but they only isolated the DNA to make THCA and had to 'feed' it CBNs to get it to make THCA... I'm hoping for the whole package...

Anybody else doing anything like this?

-Loki
 

Attachments

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TMB77

Well-Known Member
hm.

well, that miniprep procedure is just one tiny little part in a much larger procedure, depending on what it is you're trying to do with the DNA. you'll also need a PCR machine, gel electrophoresis, correct primers, a whole mess of stuff really that i'm not going to list here. I really dont get what you are attempting to do.

also that link doesnt work.
 
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TMB77

Well-Known Member
ok, made the link work. hm, interesting paper, i'm going to give it a read.

It wont be long until someone genetically modifies herb, it would probably be as simply as stably upregulating that gene to boost THC production. Stack that with some yeild enhancing genes and you could make some insane plants.
 
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Loki7

Well-Known Member
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Loki7

Well-Known Member
TMB77 said:
hm.

well, that miniprep procedure is just one tiny little part in a much larger procedure, depending on what it is you're trying to do with the DNA. you'll also need a PCR machine, gel electrophoresis, correct primers, a whole mess of stuff really that i'm not going to list here. I really dont get what you are attempting to do.

also that link doesnt work.
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I know its only one step. I was just using it as an example of how far things have come. And you don't need a PCR any more (well, I don't.. not ~need~).

I've got the restrictive enzymes and pre-cultured transfer bacterium. And I picked up one of these...

and 2 sets of gel casting trays. Along with a centifruge, magnetic stirs, tons of petri dishes, pipettes and test tubes... Custom built a clean box/incubation chamber with cold storage... I'll post some pics of it. The only pic I have right now is it in the pre fab stage...



This is what I was using for tissue cultures... Its was too small to do too much more in, hence the new model...




-Loki
 
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Loki7

Well-Known Member
TMB77 said:
ok, made the link work. hm, interesting paper, i'm going to give it a read.

It wont be long until someone genetically modifies herb, it would probably be as simply as stably upregulating that gene to boost THC production. Stack that with some yeild enhancing genes and you could make some insane plants.
Click to expand...
I'm actually hoping to transfer pot DNA into cells from another type of plant and have that plant grow something smokable with THC in it that doesn't look like a pot plant.

I want to grow outdoors. I'd have over 60 acres to use, if it didn't look like pot.

-Loki
 
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Loki7

Well-Known Member
Sensay said:
That would be awesome! Good luck. That would be a nice day job in a decked out lab.
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Sad part about that is the guy that gets that day job, probably won't smoke... Life and the cruel games she plays...

-Loki
 
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TMB77

Well-Known Member
Loki7 said:
I know its only one step. I was just using it as an example of how far things have come. And you don't need a PCR any more (well, I don't.. not ~need~).

I've got the restrictive enzymes and pre-cultured transfer bacterium. And I picked up one of these...

and 2 sets of gel casting trays. Along with a centifruge, magnetic stirs, tons of petri dishes, pipettes and test tubes... Custom built a clean box/incubation chamber with cold storage... I'll post some pics of it. The only pic I have right now is it in the pre fab stage...



This is what I was using for tissue cultures... Its was too small to do too much more in, hence the new model...




-Loki
Click to expand...

haha, nice. I'll admit man, I thought you were full of shit at first, but I see I was wrong. Nice setup you got there, you clearly have a grasp of the basic steps. I do wonder though if you have experience with plant transformation, or if you've gathered all this stuff together from doing a lot of reading and research, and are attempting to do it yourself. (which would be pretty damn impressive)

I'm curious if you could outline your plan for us, step for step. I'm fairly well versed and would love to hear the particulars of what you plan to do.

Also, admittedly my bench skills are starting to gather a bit of rust, but i'm curious how you plan to amplify the dna for a gel without using pcr...as far as I know that isnt possible. But there is always something someone doesnt know.
 
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Loki7

Well-Known Member
TMB77 said:
haha, nice. I'll admit man, I thought you were full of shit at first, but I see I was wrong. Nice setup you got there, you clearly have a grasp of the basic steps. I do wonder though if you have experience with plant transformation, or if you've gathered all this stuff together from doing a lot of reading and research, and are attempting to do it yourself. (which would be pretty damn impressive)

I'm curious if you could outline your plan for us, step for step. I'm fairly well versed and would love to hear the particulars of what you plan to do.

Also, admittedly my bench skills are starting to gather a bit of rust, but i'm curious how you plan to amplify the dna for a gel without using pcr...as far as I know that isnt possible. But there is always something someone doesnt know.
Click to expand...

This is my first attempt at DNA transformation, well my first attempt at creating my own DNA fragments. Back in the day, I did some chemistry, biology and physics... but really only the lab skills apply here. Instead of a PCR machine, I've got PCR kits... Instead of all the hassle of plasmid prep which back in the day took a day and a half, the kit does it in like 10 minutes...

So far, the hardest part was just getting the materials together. I had to get kind of creative... The stuff isn't illegal, but its hazardous... I can imagine why no one else is doing this. The technology is there though... heh, its in my freezer ;)

I don't know how much I should talk about on a public forum. Some of the materials are probably pretty easy to trace. But I'll share as much as I can.

Its pretty straight forward. What I've done so far, is grow tissue cultures of a couple strains of pot and two house plants. I created two plasmid samples from one of the pot cell cultures. One, I used 2 restriction enzymes on (just picked them out of a hat, with that article on THCA in mind). And the other I didn't do anything to. I took those and combined them with transfer bacterium. Incubated them over night and then mixed the transfer bacteriums into fresh cell cultures of the house plants. I didn't use any primers or markers, because I'm waiting on/can't get some things yet... The cultures are growing out now. This first run was mainly just getting the procedures down and test the things I had been able to get ahold of... I've been building kind of a frame to hold my camera over the electrophores gel, so I can digitize all that...

-Loki
 
beginningbotanist420

beginningbotanist420

Well-Known Member
Loki7 said:
I'm actually hoping to transfer pot DNA into cells from another type of plant and have that plant grow something smokable with THC in it that doesn't look like a pot plant.

I want to grow outdoors. I'd have over 60 acres to use, if it didn't look like pot.

-Loki
Click to expand...
when you do that, i will buy your seeds for 20$ each. i'm not kidding.
 
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TMB77

Well-Known Member
Loki7 said:
This is my first attempt at DNA transformation, well my first attempt at creating my own DNA fragments. Back in the day, I did some chemistry, biology and physics... but really only the lab skills apply here. Instead of a PCR machine, I've got PCR kits... Instead of all the hassle of plasmid prep which back in the day took a day and a half, the kit does it in like 10 minutes...

So far, the hardest part was just getting the materials together. I had to get kind of creative... The stuff isn't illegal, but its hazardous... I can imagine why no one else is doing this. The technology is there though... heh, its in my freezer ;)

I don't know how much I should talk about on a public forum. Some of the materials are probably pretty easy to trace. But I'll share as much as I can.

Its pretty straight forward. What I've done so far, is grow tissue cultures of a couple strains of pot and two house plants. I created two plasmid samples from one of the pot cell cultures. One, I used 2 restriction enzymes on (just picked them out of a hat, with that article on THCA in mind). And the other I didn't do anything to. I took those and combined them with transfer bacterium. Incubated them over night and then mixed the transfer bacteriums into fresh cell cultures of the house plants. I didn't use any primers or markers, because I'm waiting on/can't get some things yet... The cultures are growing out now. This first run was mainly just getting the procedures down and test the things I had been able to get ahold of... I've been building kind of a frame to hold my camera over the electrophores gel, so I can digitize all that...

-Loki
Click to expand...

It's a good plan to do an initial test, even without a proper construct, to get a feel for the tissue response. What sort of medium are you using in vitro?

I'd be interested to know about the construct you eventually use when you've got everything you need.

so assuming you get this construct inserted into the houseplant, confirm that with a gel of the new tissue, you'll still not know if the subsequent RNA and Protein are being produced downstream, do you have what you need to do a western blot?
 
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TMB77

Well-Known Member
and loki...even with a PCR kit, you'll still need a thermal cycler of some sort, right? how do you get around that?
 
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Loki7

Well-Known Member
TMB77 said:
and loki...even with a PCR kit, you'll still need a thermal cycler of some sort, right? how do you get around that?
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Sort of, I have to hit 70 C for 5 minutes. I was going to gloss over this part because its kinda McGyverish... Grabber hand warmers in a styrofoam cup...

-Loki
 
L

Loki7

Well-Known Member
TMB77 said:
It's a good plan to do an initial test, even without a proper construct, to get a feel for the tissue response. What sort of medium are you using in vitro?

I'd be interested to know about the construct you eventually use when you've got everything you need.

so assuming you get this construct inserted into the houseplant, confirm that with a gel of the new tissue, you'll still not know if the subsequent RNA and Protein are being produced downstream, do you have what you need to do a western blot?
Click to expand...
The pot tissue cultures are Lloyd & McCown woody plant basal w/ viatimns and a touch of sucrose... The house plant and modified cultures are a modified Gamborg basal mixture.

You are correct, I won't know until I grow the cultures out and see what they do. Right now, the gel is my only anaylsis tool. So, hopefully, I'll be able to tell that the house plant DNA has changed. That's about as much as I need to know, since I'm not sure what I'm looking for, I wouldn't be able to tell when I found it...

-Loki
 
L

Loki7

Well-Known Member
beginningbotanist420 said:
when you do that, i will buy your seeds for 20$ each. i'm not kidding.
Click to expand...
None of this is going to be for sale. If it works it'll save me a bundle of cash growing my own fields of FrankenPot... I might give it away. It probably won't work, so I'm not betting the farm on it or getting my hopes up too high. I'm having fun playing God and if something comes from it, bonus!

-Loki
 
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