How to understand the linearized plasmid backbone?

[Zhen Zhang]

Here is a paragraph in the page of 2014 iGEM Kit:

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!

[Cathal Garvey]

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

[Nathan McCorkle]

My interpretation is that the PCR leave 4bp overhangs... Insufficient for ligation... When they add the restriction enzyme the overhangs become 5bp and can undergo ligation(with another DNA strand cut with the same enzyme) :
Psti seq : CTGCA/G
Ecori seq: G/AATTC

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[Nathan McCorkle]

Linear is straight, not a loop.

Check ligation by running the samples on a gel and checking to see if the length increased.

On Jul 3, 2014 1:38 AM, "Zhen Zhang" <izg...@gmail.com> wrote:

--

[Cathal Garvey]

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!
>