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#1485758 - 01/08/09 01:34 PM
Re: Not Mothers in micro-culture
[Re: oldtree]
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Old hand
Registered: 03/31/08
Posts: 1165
Loc: Laughing Skull (US)
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Is there anything else to it other than the high pressure delivery system. It looked like an autoclave with a pressure sys. attached to it. If we looked at the mechanics of it, maybe we could build one or at the very least, knock the idea around to see if it is feasible. There's nothing too special about them. I've seen some that create a vacuum and some that use various compressed gases and even springs. The principle is the same. You have a small drop of DNA on a pellet that rides down the barrel of the gene gun when its fired. At the end of the barrel the pellet stops, but the DNA keeps going. You can buy the gold solution. Its made so that the DNA wants to chemically wrap around the gold ball, so you get a decent amount of DNA bullets... but like I said, its very crude and not worth messing with unless every other method fails. By-the-by. Where do you find bacteria cultures that you can use?, and how do you insert them? The bacteria is modified E Coli. You can get it from Bio supply places. I think I put a link in one of the previous posts, but that might have been in the Jack in the Box thread... Basically, you take your DNA and mix it with the E Coli and get it to pick up your DNA. The DNA that gets picked up by the E Coli has a gene that makes it resistant to ampicillian. So, when you culture them in ampicillian, only the transformed E Coli grow. Then you remove the cell walls of your target cells and mix them together with the E Coli. You can use either vibration in a water bath or heat shock to fuse the E Coli's plasmid in to the target plasmid. Then you get your target cells to regrow their cell wall and you're done... -Loki
_________________________
"Tastes like chicken..." - Ed Gein.
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#1485763 - 01/08/09 01:41 PM
Re: Not Mothers in micro-culture
[Re: Loki]
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Veteran
Registered: 11/21/05
Posts: 1229
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Is there anything else to it other than the high pressure delivery system. It looked like an autoclave with a pressure sys. attached to it. If we looked at the mechanics of it, maybe we could build one or at the very least, knock the idea around to see if it is feasible. There's nothing too special about them. I've seen some that create a vacuum and some that use various compressed gases and even springs. The principle is the same. You have a small drop of DNA on a pellet that rides down the barrel of the gene gun when its fired. At the end of the barrel the pellet stops, but the DNA keeps going. You can buy the gold solution. Its made so that the DNA wants to chemically wrap around the gold ball, so you get a decent amount of DNA bullets... but like I said, its very crude and not worth messing with unless every other method fails. By-the-by. Where do you find bacteria cultures that you can use?, and how do you insert them? The bacteria is modified E Coli. You can get it from Bio supply places. I think I put a link in one of the previous posts, but that might have been in the Jack in the Box thread... Basically, you take your DNA and mix it with the E Coli and get it to pick up your DNA. The DNA that gets picked up by the E Coli has a gene that makes it resistant to ampicillian. So, when you culture them in ampicillian, only the transformed E Coli grow. Then you remove the cell walls of your target cells and mix them together with the E Coli. You can use either vibration in a water bath or heat shock to fuse the E Coli's plasmid in to the target plasmid. Then you get your target cells to regrow their cell wall and you're done... -Loki wow so simple sounding. You make it seem like a 2 hours project.
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#1485790 - 01/08/09 02:43 PM
Re: Not Mothers in micro-culture
[Re: junior_grower]
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Old hand
Registered: 03/31/08
Posts: 1165
Loc: Laughing Skull (US)
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wow so simple sounding. You make it seem like a 2 hours project. Except for incubating the E Coli over night, it is about a 2 hr process... -Loki
_________________________
"Tastes like chicken..." - Ed Gein.
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#1486020 - 01/09/09 08:49 AM
Re: Not Mothers in micro-culture
[Re: junior_grower]
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Old hand
Registered: 03/31/08
Posts: 1165
Loc: Laughing Skull (US)
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Maybe just this part but what we need everybody to know is all the research and time taken before you can begin hacking. Besides the basics, you need to know the primer codes to isolate your source gene and the restrictive enzyme to cut a spot in your host DNA that will allow your source gene to bind to it. Those depend on the gene you want to transfer and the host DNA. I'm working on defining just that for all the Cannabiod genes. Once I've completed it, and done some practical testing to validate my primers, I'll post it. -Loki
_________________________
"Tastes like chicken..." - Ed Gein.
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#1486133 - 01/09/09 03:42 PM
Re: Not Mothers in micro-culture
[Re: RRog]
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Old hand
Registered: 03/31/08
Posts: 1165
Loc: Laughing Skull (US)
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Back then, recombinant DNA work was really, really new. Like working under oil lamps compared to what you're doing. Yeah, some of the stuff now a days is just out of control. You can get nucleotides engineered with fluorescent molecules attached to them. So when you build your rDNA with them, under a microscope and UV, you can see your bit of DNA glow inside the cell. -Loki
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"Tastes like chicken..." - Ed Gein.
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