Re: [DIYbio] PEG transformation - plasmid size

Recombination in E.coli depends on the strain, the plasmid, and the
plasmid content. If you're using recA- strains, which are lacking a
recombinase gene, then DNA is generally pretty stable. If you're using a
plasmid that isn't usually stable with large inserts, then you can
expect trouble. And, the larger the insert, the more opportunity there
is for the plasmid to find suitable sites for recombination that lead to
deletions; that's just plain statistics, but it mightn't be significant
enough to worry about.

You can reduce your odds of gene loss by designing your gene to include
plasmid or gene maintenance stuff; you've been looking at
toxin/antitoxin systems in another thread, you could make those part of
your gene cassette, possibly even part of the operon you're designing.
If you're synthesising a plasmid from scratch, you could even include
your resistance marker (if any) in the operon, too.

Regarding Agrobacterium transformation, Sebastian would know more. I
think you can do E.coli-style heatshock on Agrobacterium, I haven't
heard of transformation using Nitrogen before..

On 03.02.2013 12:14, Andreas Sturm wrote:
> Thanks for your help!! 
>
> And about recombination deletion or mutation, is this an issue? I
> mean if there is no genomic E coli DNA. Copy number is high. 
>
> You mean the heat shock with liquid nitrogen for agrobacterium? Or
> the usual E.Coli procedure for agrobacterium - never heared that
> would
> work... ? 
>
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