Agrobacterium tumefaciens - how to get, and how to transform? - Advanced Mycology

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Feelers
Anti-Myth-Rhythm-Rock-Shocker

Registered: 06/18/02
Posts: 1,806
Loc: Land of Oz
Last seen: 5 years, 11 months

Agrobacterium tumefaciens - how to get, and how to transform?

#13149695 - 09/05/10 08:15 AM (13 years, 7 months ago)

Hi guys - I'm looking to sort my procedures out well in advance, and have decided on Agro T to do my Ps. cubensis transformation. I'm ok at genetics, but I've never seen this done in person before. :eek:

1. First up - where can I get a lab strain of Agro and what one should I use - can you simply buy this online? I might ring the university round here and see if they'll just give me some.

2. Does anyone know of a good source to tell you how to make/design the plasmids? What restriction enzymes/antibiotic resistances to use etc, how to put the genes in the correct order. I'm not sure whether I need to bother with all the E coli swapping plasmid stuff - or would this route be a better idea.

3. I've been looking into how to actually get the Ti plasmid into the cells, and have found two options so far - electroporation and the "Freeze-Thaw Method". Electroporation needs an electroporator obviously, but the Freeze thaw requires Liquid Nitrogen. I'd rather own an electroporator - but they don't seem cheap, if anyone has some ideas on this - DIY plans or where to get second hand ones that would be excellent. Alternatively where can you get Liquid N2 easily?

Does anyone here work with this sort of thing?

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solumvita
Q.B.E.

Registered: 02/12/08
Posts: 2,061
Loc: South Africa
Last seen: 10 months, 4 days

Re: Agrobacterium tumefaciens - how to get, and how to transform?

[Re: Feelers]
#13170868 - 09/09/10 02:40 PM (13 years, 7 months ago)

FastFred is the guy to answer those questions

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