Metacelsus
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Good places to start learning about making recombinant organisms?
I'm interested in techniques of making recombinant organisms, likely starting with bacteria. (I know much of the theory already). Any suggestions of
where to start learning?
For example, say I want to insert a gene for ampicillin resistance into E. Coli. How would I make a plasmid with that gene (with restriction enzymes
and DNA ligase), and what would the steps be to get it into the cell?
[Edited on 3-7-2015 by Cheddite Cheese]
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mayko
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Here's a lab manual for a relatively advanced genetic engineering course. I also have in harcopy a more basic lab manual for molecular biology, which
includes protocols for simple transgenics like GFP in E. coli, and I could scan parts of it.
As far as supplies go, I think that Carolina Biologicals has a decent inventory and generally a good reputation for being amateur-friendly (though some of their other stock is
regulated as agricultural pests or low-level biohazards).
[Edited on 3-7-2015 by mayko]
dang, PDF is greater than 10MB. Here's an online version:
Genetic Engineering Lab Manual
[Edited on 3-7-2015 by mayko]
al-khemie is not a terrorist organization
"Chemicals, chemicals... I need chemicals!" - George Hayduke
"Wubbalubba dub-dub!" - Rick Sanchez
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crazyboy
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OpenWetWare has lots of procedures with lots of detail: http://openwetware.org/wiki/Protocols
This is something I've been interested in for a while. It seems like despite interest from hobbyists such as yourself and others DIY molecular biology
is much harder than DIY electronics or chemistry. I think the limiting factor here is the cost of equipment and reagents. You'll probably need a
centrifuge, a PCR machine, gel electrophoresis equipment, micropipettes, buffers, restriction enzymes, ligase, an incubator etc.
It isn't out of the hobbyist range but I do think it will cost a fair bit. Some of the equipment can be bought used at a reasonable price and some
plasmids or bacterial strains you might be able to get from a friendly researcher. Some stuff though I'm really not sure about. For example I have
inserted genes into E. coli, collecting the E. coli cells from the culture media required centrifugation of ~50ml of solution that required a large
and very expensive centrifuge. Also bacterial cultures are generally stored at -80C which is much colder than a household freezer.
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Tsjerk
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I think you can find relatively cheap alternatives to most of the expensive stuff. For example 3 water baths (and a lot of patience) instead of a PCR
machine. Gel electrophoresis doesn't require more than a plastic holder and a power supply. Micropipettes can be bought second hand. Instead of
centrifuging you could filter your bacteria etc.
Only the enzymes are going to be a problem I think...
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PHILOU Zrealone
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Lonza electroporation transfection nucleofection
I have worked there and I have learned that it is very efficient way to introduce exogen DNA/RNA into living organic cells without use of expensive
viral technologies (viruses vectors, special accredited lab to work with); those are also often ineffective with specific cells and are quite time
consuming.
Here the procedure is fast, cheap and effective.
You have a witness DNA coding for a green fluorescent protein that is visible/expressed 20 minutes after transfection.
This allows one to fine tune the transfection condition to the specificity of the living cell in a way to acheive the best transfection yield (pulse
intensity (voltage), pulse shape, salinity, pH, viscosity, solvent, T°,...)
You may even make multiple transfection in one shot; or after stabilisation of a specific transfected line of cells, you can redo transfection with a
second DNA strand...
There are two devices types and electrodes types, one for floating cells and one for adherent cells (cells that needs a support to grow).  
[Edited on 3-7-2015 by PHILOU Zrealone]
PH Z (PHILOU Zrealone)
"Physic is all what never works; Chemistry is all what stinks and explodes!"-"Life that deadly disease, sexually transmitted."(W.Allen)
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crazyboy
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Quote: Originally posted by Tsjerk  | I think you can find relatively cheap alternatives to most of the expensive stuff. For example 3 water baths (and a lot of patience) instead of a PCR
machine. Gel electrophoresis doesn't require more than a plastic holder and a power supply. Micropipettes can be bought second hand. Instead of
centrifuging you could filter your bacteria etc.
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I get what you're saying but I think this is only true to an extent. Yes PCR was once done with three water baths, but computation was once done with
vacuum tubes it's still obsolete. I've never heard of filtration as an alternative to centrifugation, I understand what you're getting at but I'm
skeptical. Again I don't think it's out of reach after all many people have sound systems or home theaters or woodshops with tens of thousands of
dollars invested.
| Quote: |
Lonza electroporation transfection nucleofection
I have worked there and I have learned that it is very efficient way to introduce exogen DNA/RNA into living organic cells without use of expensive
viral technologies (viruses vectors, special accredited lab to work with); those are also often ineffective with specific cells and are quite time
consuming.
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This is interesting but not particularity helpful I think. I'll bet those units aren't cheap, and who is talking about using viral vectors? Making
cells competent with calcium chloride and transfecting with heat shock may not be the most modern or efficient technique but it works and it's dead
simple.
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smaerd
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A lot of the equipment can be had cheaply if one is crafty. Ex:
PCR - http://www.instructables.com/id/Arduino-PCR-thermal-cycler-f...
Gel electrophoresis - http://teach.genetics.utah.edu/content/build_gel_box.pdf
Centrifuge - http://www.instructables.com/id/OpenFuge/
Gene Gun - https://www.youtube.com/watch?v=Dw5MJHh86Fc
And things like subsituting autoclaves for pressure cookers. Although autoclaves are available second hand. Who really wants one in their house
though? Not me. I am eerie of almost all second hand bio stuff.
Some of the more serious biotech equipment is of course no joke expensive.
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phlogiston
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Equipment is not the biggest problem. Much of it can be made yourself or improvised.
A much bigger problem is that is very difficult to get supplies, and they are expensive. An nice stock of restriction enzymes will set you back a few
thousand EUR (or USD). PCR supplies cost hundreds of EUR/USD for just tens of reactions. You burn money like water if you do some serious
experimenting, and all of these things have a limited shelf life too.
Also, lack of interesting colours and explosions or other clearly visible results may be another reason amateur scientists tend to prefer other areas
of chemistry.
-----
"If a rocket goes up, who cares where it comes down, that's not my concern said Wernher von Braun" - Tom Lehrer |
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crazyboy
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This mentality is part of the problem, this stuff is crap. That homebrew PCR requires a machine shop to make the sample holder, that centrifuge costs
$200 and requires a laser cutter, the electrophoresis box is just a waste of time.
For only a little more you can get working, legitimate, QUALITY equipment. See:
http://www.ebay.com/itm/Beckman-Coulter-Microfuge-18-Centrif...
http://www.ebay.com/itm/Thermo-Hybaid-PCR-Express-Thermal-Cy...
http://www.ebay.com/itm/PERKIN-ELMER-Cetus-480-Variable-Temp...
http://www.ebay.com/itm/Perkin-Elmer-GeneAmp-PCR-System-2400...
For $250 you can get a Beckman centrifuge that goes 300% of the speed, hold 600% of the samples, for 25% more in cost than the "OpenFuge".
You can get a PCR that 12 times as many samples for LESS MONEY than the "OpenPCR".
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phlogiston
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Not even that long ago, a few decades, every lab made their own electrophoresis equipment and people used alternating warm and cold water baths to do
PCR. Sure, it is inconvenient and you have a lower chance of success/reproducability, but for a while that was state-of-the-art and could still be a
reasonable methods for amateur scientist.
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"If a rocket goes up, who cares where it comes down, that's not my concern said Wernher von Braun" - Tom Lehrer |
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crazyboy
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| Quote: Originally posted by phlogiston | | Not even that long ago, a few decades, every lab made their own electrophoresis equipment and people used alternating warm and cold water baths to do
PCR. Sure, it is inconvenient and you have a lower chance of success/reproducability, but for a while that was state-of-the-art and could still be a
reasonable methods for amateur scientist. |
Yes you're right, all I'm saying is I think we're past that point with the amount of surplus lab equipment in the market. I do agree with you about
the cost of reagents as well it's not cheap.
The way I see it there are really three options if you're serious about this.
1. Invest the money yourself and get a legit bio lab. I don't think this is unrealistic if you have a solid disposable income and this is your primary
hobby. That said it most hobbyists don't want to shell out all that cash for what they see as making some glowing bacteria.
2. Join a bio hacker space. Now I've never been to one personally but there are places especially in the bay area like BioCurious and Counter Culture
Labs. These places charge a membership fee but they have most of the equipment you could want and probably some reagents as well in addition to other
interested hobbyists.
3. Find a university professor that is willing to help you out or let you use their equipment or work at a biotech company and use their stuff during
the down time.
I think #2 is the most reasonable, Counter Culture Labs had an iGEM entry to make milk proteins from yeast to produce real vegan cheese. They also
raised $33,000 to upgrade their lab space which is pretty impressive.
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Metacelsus
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I've done some more research, and this phrase is surprisingly, infuriatingly, common:
| Quote: | | This item is sold and shipped to schools and businesses only. Not available to individuals or residential addresses. |
Any good ideas of where I can get materials (specifically, restriction enzymes?).
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crazyboy
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Have you tried New England BioLabs? https://www.neb.com/ I've never tried ordering from them as an individual but nothing in their terms of service or shipping explicitly mentions
only selling to business.
Also I have ordered products and claimed to be a business, sometimes it isn't necessary and sometimes it isn't sufficient but it rarely hurts.
[Edited on 17-7-2015 by crazyboy]
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