Thanks for the info!!
Well, I saw that there were *loads* of electroporation cuvettes in a proffessor's lab, so we 99% have one.
But, for easy repeating, we'd like to try PEG transformation first... It works with all kind of cells... In case it doesn't work with PEG, we try it with e.poration the.
On Wed, Dec 19, 2012 at 5:58 PM, xmort <moravec@ueb.cas.cz> wrote:
Electroporator will be necessary for direct transformation of ligation mixture to Agro. If it is available at the university you should be OK. However I would experiment with direct agro transformation only if it really shows great instability in E.coli. It might be and should be OK though.
Dne úterý, 18. prosince 2012 16:39:56 UTC+1 Mega napsal(a):Sounds great, thanks!
Oh, 800 bp is quite a big potential for recombination...On Tue, Dec 18, 2012 at 10:19 AM, xmort <mor...@ueb.cas.cz> wrote:
. There is an "undocumented" insert of about 800 bp between SaRep and Kan selection marker. This is E-coli genomic DNA, so it might increase the tendency to recombine with genomic DNA in E.coli. Also Kan marker might be in opposite orientation than some of the maps show. This might be important,
But I don't think we can directly transform it into agrobacterium with the PEG transformation methode because efficiency will be quite low...? On the other hand, if we "just" get three transformed colonies, we have three colonies, of which one may have the insert.
Or we get the electroporator which is in one of my university's labs, but this was kind of plan B.
However, we could try it. The betaglucosidase on X-Gal will show where the insert is, it may work in agrobacterium too (gram negative)...
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