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

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Honestly, that block of text seems a bit mangled and I'm not sure if the
correct meaning is clear. Maybe I just need more coffee.

The reasoning, though, is that it's *really easy* to produce *lots and
lots* of plasmid backbone using PCR, which is what the iGEM guys want;
loads of DNA at minimal cost and effort. So, PCR is great. But, PCR
makes linear DNA, and there are a few considerations when working with
linear DNA; if you want to cut it at the ends with restriction enzymes,
there must be a little bit of extra DNA for the enzyme to "land" on
before it cuts the ends.

From there, it's normal ligation procedure, but when amplifying a
plasmid at RE-sites, it's normal to add a few extra nucleotides to your
primers to let the reaction proceed, I'm assuming that's part of what
the block of text was trying to relate.

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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