Re: [DIYbio] Bioluminescent yogurt (Again!)

On Tue, Apr 3, 2012 at 2:34 AM, elegans <tashant@gmail.com> wrote:
> "For the linear transfer, I am scared if the exonucleases would just chew of
> the construct. Is there any literature on how to avoid that, also won't the
> low temperature in ice bath inactivate the enzymes?
>
> Thanks,
> Harsh"
>
> Yes, exonuclease activity is required for recombination to occur. The viral
> beta protein will protect the single stranded fragment and promote insertion
> of the construct as an okazaki fragment. Here is a good recombineering guide
> from Stewart's lab.

not sure why you keep mentioning the requirement of viruses... clearly
NEB is making $ and happy customers with their virus-free kit to
transform K. lactis with linear DNA via homologous recombination:
http://www.neb.com/nebecomm/products/producte1000.asp

>
> http://www.biotec.tu-dresden.de/research/stewart/group-page/recombineering-guide.html
>
>
>>
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--
Nathan McCorkle
Rochester Institute of Technology
College of Science, Biotechnology/Bioinformatics

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