http://fivephoton.com/pdfs/Calcium%20Phosphate%20Transfection%20Kit.pdf
An actual experimental procedure!

Helgoland

How the hell do they get them to you frozen, then?

They are probably shipped in insulated containers with dry ice and cold packs packed inside These type of packages can remain cold for a few days

Thanks, lugh.  I never was quite sure.

Vesp, from my understanding lots of plasmids could be relevant to your interests, depending on what you're splicing and what kind of cells you are transforming.

Take a look for instance at pBR322
http://en.wikipedia.org/wiki/PBR322

It used to be one of the most commonly used plasmids with e. coli and is still widely used.  It has instructions coding for reproduction and for ampicillin and tetracycline resistance genes, as well as unique sites for more than 40 different restriction enzymes.

So if you are using EcoRI, EcoRV, etc. to cut out the bit of dna that interests you, you can easily splice it into pBR322 because there is only one place on it that enzyme cuts.


I've been reading and watching quite a few videos and one thing that isn't particularly clear and seems to get glossed over is how to get the particular piece of DNA you want after cutting something complicated like plant or animal DNA.  Any enzyme you use is going to cut it in multiple places but you only want one of the pieces.

The tail end of one video (I can't remember which) suggested that plasmids are spliced with the mixed DNA pieces, bacteria transformed and cultured, then each culture tested to see if it's making the particular protein in which we're interested, but that seems like a mess.

You should know the length of the DNA piece you want, isn't there some way to separate it out via centrifuge or electrophoresis after cutting and before splicing?

http://www.wiley.com/college/pratt/0471393878/student/animations/dna_sequencing/index.html
undergrad type stuff on sequencing, gotta understand the process
Electrophoresis might be useful for the initial separation, but if you want pure dsDNA, you'll be using chromatography.
If you've got access to a good university library, check out the book DNA Chromatography by Gjerde & Hanna.  It's everything you'll need to know.  Some of the techniques can be applied on a less-than-institutional level with a little imagination.

This place is great for finding media preps among other miscellanous information for most strains.  I also believe you can order strains from them, however I am yet to go that route.  http://www.dsmz.de/dsmz/main.php?menu_id=2

My favorite site for biotechnology research currently is http://www.brenda-enzymes.org/ .  Is an enzyme database... great for finding enzymes and which microbial critters they are found in.  Currently doing research on phenylalanine decarboxylase from lactobacillus brevis currently.  Essentially kicks out phenethylamines.

Seriously though applying retrosynthetic approach with that enzyme database gives an interesting approach to developing useful synthetic routes utilizing microbial transformations.

And PCR in your kitchen! :p  http://www.scientificamerican.com/article.cfm?id=pcr-at-home

Thank you for the link, it does look useful and is probably one of the most advanced and largest forums on the net when it comes to genetic modification and general biology that relates.

You say DIY bio is a small passion of yours? What sort of stuff have you done in the past/know the most about? It would be greatly appreciated if you took the time to share it with this forum! I would love to see the biology section of this site grow, as I think it is always an excellent complimentary subject to the sites main topics.