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 I need your help! 
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Joined: Thu Jun 25, 2009 12:12 am
Posts: 22
Post I need your help!
Okay this has been bugging me, how do the people from igem and other things make DNA or read DNA and I do not mean RNA. Whenever I search I do not find anything, is it like a person has to go to these big universities. Also where do I order synthetic DNA and were if I have to where do I get the know how to do this all.

Sorry the lack of information on the internet is irritating, if you guys star moving of topic I’ll remind. Sorry I just want to get to the botum of this splicer work your magic.


Thu Dec 10, 2009 5:55 am
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Joined: Fri Feb 29, 2008 5:26 am
Posts: 56
Post Re: I need your help!
Students and professors have the easiest access to a larger selection of products. Outside of biotech companies, there pretty much the only other demographic that's ordering biological supplies. In alot of cases, these people have to register for accounts on the sites they wish to purchase supplies from. I've seen sites that require students to list their university and professors for verification. As far as general citizens are concerned, companies probably see it as a liability issue. It's probably tougher for a company to verify that you know what your doing with the more dangerous supplies and that you know how to handle them safely.

Is there any particular reason you want synthetic DNA? I know there are plenty of companies that can create custom sequences.

As far as the whole "where do I start" check the forum. There are plenty of free documents online. Also check out your library or local used bookstore.


Sun Dec 13, 2009 7:07 am
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Joined: Sat Apr 04, 2009 7:51 pm
Posts: 25
Post Re: I need your help!
Teslaboypunk wrote:
...how do the people from igem and other things make DNA or read DNA and I do not mean RNA.


It depends on how you want to make it, there's actually couple of different ways.

You can for example make DNA by chemical synthesis. In this way different nucleotides are joined together in sequences. By chemical synthesis it is possible to create any sequence wanted, but the limiting factor is the lenght of the synthetised sequence due to the relatively frequent errors.
more of this here: http://en.wikipedia.org/wiki/Oligonucleotide_synthesis

Second way would be to use PCR, but for this approach you need a template sequence that is then copied. For a template sequence you can use basically any other DNA sequence even ones synthesized chemically. This way it is actually possible to synthesize also longer DNA sequences by combining chemically synthesized oligonucleotide primers and templates and PCR or ligating enzymes.
More here: http://en.wikipedia.org/wiki/PCR
and here: http://en.wikipedia.org/wiki/Gene_synthesis

The easiest and most reliable way to multiply DNA, if you just want to multiply it not edit it anyhow, is to just grow it in suitable bacteria (E.coli) as a plasmid. This is basically the routine way of cloning DNA and if you do DNA work you always end up in cloning the wanted product into a plasmid that you can then maintain in bacteria. And if you have suitable restriction sites in your plasmid the easiest way of editing DNA is to use restriction enzymes and just cutting and pasting pieces of DNA together.
Some more in here: http://en.wikipedia.org/wiki/Recombinant_DNA

In iGems constructs I'd say that they have cloned the different parts with PCR and added restriction sites around the genetic elements (genes, promoters, etc.) and then just cloned the parts into suitable plasmids. The plasmids are then just grown in E.coli and purified.

Dr. Wikipedia answers :)

Teslaboypunk wrote:
Also where do I order synthetic DNA


I needed to edit this answer because I stumbled upon these links to different firms that sell synthetic DNA and basically anyone can order it (I suppose).

DNA2.0
GeneScript
Eurofins MWG Operon
Geneart


Last edited by ATG on Sat May 22, 2010 12:49 am, edited 1 time in total.

Tue Dec 15, 2009 12:16 am
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Joined: Tue Sep 29, 2009 4:39 pm
Posts: 21
Location: Hungary
Post Re: I need your help!
Peopleeeeee! I need help! I want to transform plants but I cant get my hands on agrobacter culture... If I just shoot like a GFP plasmid into a plant cell with something like a hommade gene gun would it work? What do you think?

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Mon Jan 25, 2010 4:28 pm
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Joined: Sat Apr 04, 2009 7:51 pm
Posts: 25
Post Re: I need your help!
I'm interested in how you would make a DIY gene gun.

I was watching this how to use a gene gun introductoy video and wondering if a bb-gun could be used as one. I'm not sure, but I'd expect the pressure levels to be high enough.


Tue Jan 26, 2010 12:53 am
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Joined: Mon Jun 01, 2009 4:34 pm
Posts: 60
Post Re: I need your help!
You could always up the pressure levels if you had to anyways, but your biggest problems with a homemade DNA gun would be the vacuum, maintaining sterility, and making sure the little busting caps (I forget what they're called) fit.

DNA guns should be tools of last resort, though; A. tumefaciens etc. work better.


Sun Apr 04, 2010 2:48 pm
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Joined: Sun Dec 27, 2009 7:57 pm
Posts: 15
Post Re: I need your help!
I wouldn't recommend using a home made gene gun, additionally, you should try and get specifications to the off the shelves one before considering building one yourself. you might find it hard to replicate.


Wed May 19, 2010 1:07 pm
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Joined: Thu May 20, 2010 8:02 am
Posts: 2
Post Re: I need your help!
You can order synthetic DNA constructs from DNA2.0 https://www.dna20.com/ though I suspect is it quite expensive.

Particle bombardment does work for making transgenic plants though it is rather labor intensive as it requires in vitro plant cell culture and the use of selectible markers such as antibiotic or herbicide resistance. Agrobacterium is difficult to get your hands on and would be hard to work with without access to an autoclave, electroporator lots of antibiotics and such.

Depending upon what your goals are a homemade gene gun wouldn't be too difficult to make workable. Major plant bio labs have and use homemade gene guns. The first were based around actual guns nowadays they use compressed helium, a vacuum pump, a solenoid valve and some DNA precipitated on gold particles on a piece of aluminum foil a cartridge over a vacuum chamber. There is a paper describing plans that I could probably find for you. The trick is that transformation of individual cells is all that is achieved with the gene gun, then you have to regenerate a fertile plant from the cells transformed with the gene gun. Getting that to work, especially with a new species of plants can be a whole scientific career.

To see the expression of a transgene in a leaf or onion peel, gene guns work great.


Thu May 20, 2010 8:28 am
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Joined: Mon Jun 01, 2009 4:34 pm
Posts: 60
Post Re: I need your help!
9 times out of 10 you can replace an autoclave with an alcohol bath and a trip through a pressure cooker. The issue that that doesn't address, though, is nuclease contamination; you really need a MiiliQ or similar filter device to ensure success.


Sun Jun 13, 2010 4:40 pm
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Joined: Fri Oct 08, 2010 6:08 am
Posts: 58
Post Re: I need your help!
Great video from wired.


Fri Oct 08, 2010 6:20 am
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