Re: [DIYbio] How to understand the linearized plasmid backbone?

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FYI, in off-list correspondance I learned that the intention of the
passage was to say that, once cleaved, the small fragments at either end
of the linear plasmid are unlikely to compete for annealing-space with
an insert. Reading it again, this is clear, but for some reason evaded
my brain out of context (I'm inclined to blame sleep-dep).

I was also reminded that one other feature of PCR-product linear plasmid
is that the DNA is unmethylated, meaning you can use
methylation-sensitive enzymes to dice up the template plasmid, leaving
*only* linear vector and reducing background transformants with the
original vector later on.

On 03/07/14 09:38, Zhen Zhang wrote:
> Here is a paragraph in the page of 2014 iGEM Kit
> <http://parts.igem.org/Help:Protocols/Linearized_Plasmid_Backbones>:
>
> Why Linearized Plasmid Backbones?
>
> Short single stranded DNA fragments will not ligate to 4 bp overhangs. By
> creating a very short overhang on a PCR of a plasmid backbone, the remnant,
> when cut with EcoRI and PstI is sufficiently short that it will not anneal
> at ligation temperature, and will therefore not ligate. This allows us to
> build high quality construction plasmid backbone without purifying away the
> cut fragments remaining after PCR.
> -----------------------------
>
> And is the linearized one is still a *loop-like one? *What is the different
> between the usual one and this one? And how to check if the parts are
> ligated on the backbone?
>
> Thank you for your help!
>

--
T: @onetruecathal, @IndieBBDNA
P: +353876363185
W: http://indiebiotech.com

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