RF-Cloning.org Forum

Anything and everything cloning: Go...

You are not logged in.

#1 2014-08-11 18:40:16

Sand
Member
Registered: 2014-07-25
Posts: 11

Question on the basics

Dear ,


I have the following question:
In a paper (see below for the link) they mention the following: "A new product strand does not contain a binding site for the reverse megaprimer and is therefore not a template for the next round of PCR, causing a linear rather than exponential amplification"

I understand it as: the generated new strand (from 5-3') , upper part in the picture about RF cloning, can not be used as a template because it contains a nick and thus the primers wont work.
This I understand (unless I am wrong here?)
But the last part of the second picture about RF cloning in picture one is a bit fuzzy for me.
What do they mean with the + "red circle" part?


Do they just mean that "bleu template"(formed by the PCR) anneals with the red part to form the ds plasmid?

But where does the nick in the red part come from?

Is this because of the red template being formed based on the bleu parental template?



See this link for the picture: http://www.plosone.org/article/info%3Ad … ne.0053360
(picture one, at the bottom, upper part and lower part)

Offline

#2 2014-08-11 19:29:20

Steve Bond
Administrator
Registered: 2014-01-23
Posts: 126

Re: Question on the basics

Hey Sand,
You're probably more or less on track regarding the first bit, but instead of thinking of it as a nicked plasmid, just look at is as a single linear daughter product. When the reverse complement mega-primer binds, it's 3' end is situated right at the terminus of the strand, so it can't initiate any further synthesis. Thus the linear expansion in rf-cloning vs. exponential of normal PCR.
That little + red thingy is just trying to convey that the reverse complement of the plasmid was still synthesized from a parental source by the other half of the mega-primer, and when you combine the two daughter strands, you get a full plasmid with a pair of nicks. They should have changed the colour of the incoming daughter strand to green or something, because it really looks like the parental plasmid acquired a nick somehow (it doesn't).
Does that clarify, or am I just muddying the water further?
-Steve

Offline

#3 2014-08-11 19:49:27

Sand
Member
Registered: 2014-07-25
Posts: 11

Re: Question on the basics

beako1980 wrote:

Hey Sand,
You're probably more or less on track regarding the first bit, but instead of thinking of it as a nicked plasmid, just look at is as a single linear daughter product. When the reverse complement mega-primer binds, it's 3' end is situated right at the terminus of the strand, so it can't initiate any further synthesis. Thus the linear expansion in rf-cloning vs. exponential of normal PCR.
That little + red thingy is just trying to convey that the reverse complement of the plasmid was still synthesized from a parental source by the other half of the mega-primer, and when you combine the two daughter strands, you get a full plasmid with a pair of nicks. They should have changed the colour of the incoming daughter strand to green or something, because it really looks like the parental plasmid acquired a nick somehow (it doesn't).
Does that clarify, or am I just muddying the water further?
-Steve

Yes that is exactly what I ment!
Perhaps I used to wrong words to call it a "nicked plasmid strand", I did mean what you said: a single linear daughter product that can not be amplified because its linear (there is a "hole" between the two "ends" that should "anneal" to form the full plasmid).

I was on the right track! The colors should indeed be changed because now its confusing and it seems that the "red part" is somehow created even of the primer can not bind... Its confusing and did not make sense.

Offline

#4 2014-08-11 20:08:05

Steve Bond
Administrator
Registered: 2014-01-23
Posts: 126

Re: Question on the basics

Cool cool, good luck with your project.
-Steve

Offline

Board footer

Powered by FluxBB