I'm totally new please help

[QDragon Leet]

Hope this isn't annoying but i literally had no idea this kind of DIY biotech was possible. I have wanted to do this kind of thing for years. What i'd really like is any clear guides to getting started, particularly in actually growing microbes and implanting new DNA. (some light googling was surprisingly lacking of such resources) One issue is that have a severe physical disability, so i'm going to need lots of help but i've got ideas!

Sidenote: any members from vancouver island?

To sum everything up, links and lots of them. For guides, suppliers, other forums, anything and everything. Also please contact me directly if you like i'm extremely excited to get involved.

Thanks, Q

[Derek]

Hi Q,

I'm in Victoria, BC. I've got a space in Rock Bay Square that we use for classes and projects, biospace.ca. Feel free to contact me off-list.

--Derek

[QDragon Leet]

wow so glad there's a lab on the island 

please pardon my ignorance but how exactly do i contact you directly? i can't click on your name 

[Nathan McCorkle]

Here's a good place to start:
http://openwetware.org/wiki/DIYbio/FAQ

You've basically got to learn the lingo/terminology to effectively do
google searches. If you post the searches you tried making, I can try
and help you restructure them in a way that will get you lots of
results (often too many results IMO)

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Nathan McCorkle
Rochester Institute of Technology
College of Science, Biotechnology/Bioinformatics

[QDragon Leet]

Thanks, what i don't understand and would like to is how exactly a DIYer introduces a new gene into an organism? I always thought that process was extremely complicated especially when linking the new gene to be triggered by an existing 1.

[Mega]

I always  recommend watching this video.
http://www.youtube.com/watch?NR=1&feature=endscreen&v=sovDqqhQJx8

You'll get a good overview with how less you can insert DNA (plasmids) into bacteria.

[Mega]

May I add:

With bacteria, it's  not at all very difficult to insert a new gene.

Plasmids are mobile elements, which the bacteria can easily take up.

Of course, plasmid stability is another issue, but it's good to know as a beginner. And for gene coloning. Plasmids are amazing! ;)

Am Samstag, 12. Mai 2012 07:54:11 UTC+2 schrieb QDragon Leet:

[tardigrade]

There are a few ways:

By far the easiest:

You can stick the gene onto a plasmid and then transform it into bacteria or transfect it into eukaryotes.  If the gene has an appropriate promoter its transcription can be activated by other genes.

Usually an antibiotic is used to keep the plasmid within the bacteria or eukaryotic cell, assuming the plasmid confers resistance to that particular antibiotic (if it doesn't, then adding the antibiotic will kill the cells).

See "stable and transient transfection": http://en.wikipedia.org/wiki/Transfection#Stable_and_transient_transfection , and "yeast integrative plasmid".  Various companies make plasmids specifically for these purposes.

-----

Various viruses can also be generated which will express a gene of interest.  Often these viruses will quickly kill the cells, so this is used mostly to bulk up protein (google "baculovirus expression" for an example).

Some viruses are crippled so they cannot reproduce and kill cells; this is one of the mechanisms of gene therapy (I think one of them is a crippled form of "adeno-associated virus", but I've never used it).

-----

In the lab I work in at school we use homologous recombination (utilizing "selectable markers" to screen for recombinants) to knock out certain genes.  Following knockout of a gene with a selectable marker it is then possible to knock-in a replacement gene and counter-select by looking for transformants which no longer grow in the presence of the selection.  PCR with primers flanking the region of interest, and primers internal to the knocked in gene, can be used to verify the gene successfully integrated exactly where you wanted it to.

http://openwetware.org/wiki/CH391L/S12/Selectablegeneticmarkers

http://en.wikipedia.org/wiki/Gene_targeting 

I'm sure there are more ways of integrating genes, but these are the ones I can think of now.

The big issue in all of them is ensuring that you don't disrupt important parts of the DNA when integrating the foreign DNA.  There are various methods to verify this (Southern blots, Inverse-PCR and variants, probably more).

All of this is significantly easier now that the sequences for multiple genomes are available.