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#1 rf-cloning troubles » DpnI » 2015-01-24 19:49:03

Sand
Replies: 1

What do you all think about the DpnI treatment for EMP cloning and/or RAM cloning?
In the RF cloning, I can see the point since its a linear amplification, but in EMP and RAM cloning you get an exponential amplification of the plasmid. I wonder of the parental plasmid needs to be destroyed in the end when you have a lot more amplified plasmid.
Any ideas on the necessity of the DpnI treatment/efficiency?

#2 Re: rf-cloning troubles » Is phosphorilation needed? » 2015-01-17 20:05:20

Steve Bond wrote:

Nah, if you just transform bacteria with linear DNA, they chew it up. Wether you use pre-phosphorylated primers or T4 kinase, you will need to include a ligation step before transformation.
As far as using homologous overhangs on the primers, I'm not actually sure what you mean. If you add any extra sequence to the ends of the primers for EMP, it will be amplified along with everything else and you'll still be left with blunt ends.

Yes: blunt ends indeed, but they would be homologous to eachother!

What I was thinking is this: when you desing the two new primers (for the secondary PCR in EMP) you could make them so that both 5' ends are homologous to eachother. If they would be homologous for a few basepairs (I dunno 10 to 20 perhaps) would the bacterium not be able to link them using some sort of homologous end joining?
I am not sure you see what I mean. Perhaps I should try to draw it.

perhaps this will help:

5' NNNN ______________ NNNNN 3'
3' NNNN ______________NNNNN 5'

N-parts : are the parts that are homologous because you created the primers (for the second reaction) in such a way that they overlap that the 5' part (so after the PCR, you have this kind of DNA with the N-parts).


I see it more as some sort of "double strand break" in wich the bacterium thinks this is a double strand break and it will "recombinate" them again?

Is this clear?

#3 Re: rf-cloning troubles » Is phosphorilation needed? » 2015-01-17 19:25:26

Steve Bond wrote:

My pleasure smile

I wonder: if they do the EMP, why would they now use phosphorylated primers? It seems that this might do the trick to form a plasmid again!
A bacterium should be able to have a recircularization I think or perhaps this is a bit far fetched!
I wonder what if you used homologous overhangs on your primers when doing this: both ends would be homologous, I can imagine that it would be easier to have a recircularization then!

#4 Re: rf-cloning troubles » Is phosphorilation needed? » 2015-01-17 18:56:37

Steve Bond wrote:

Ha, well... I think the term 'Spherical Force' may sound a bit more mystical than it really is. Here, I've drawn a picture:

http://www.rf-cloning.org/fluxbb/img/po … loning.png

Does this help?

And yes, the amount of starting material is rather important. This is not your run-of-the-mill PCR reaction.

-Steve

Ah yes, now I see it.
I should have drawn everything to see it visually!
Thanks.

#5 Re: rf-cloning troubles » Is phosphorilation needed? » 2015-01-17 17:56:16

Steve Bond wrote:

You are right: when the parental plasmid denatures, it stays a circle, but when the daughter plasmids denature they become linear. The reason the daughter products re-anneal into circles again is because the relative positions of the 5' and 3' ends of the sense and antisense strands are offset by the length of the mega-primer; this leads to a nicked, circular product.
With the EMP process, the newly synthesized sense and antisense strands are perfectly matched up, and the PCR is designed to work in a very canonical fashion. That's why blunt end ligation is necessary in EMP.
Maybe it will help to think about how the insert behaves during each method. During 'normal' RF-Cloning, the insert itself is not copied at any point during the 2° PCR; it is simply a passenger component of the mega-primer. In the EMP method, the insert is copied along with the rest of the plasmid.
Does that make any more sense?
-Steve

So somehow, the fact that the "insert" (the megaprimer, except the "binding parts of the primer") is not copied a circle is formed. So it has to do with some sort of "spherical force" attributed by this megaprimer (insert)?
I see what you mean (I think), but find it hard to understand why this leads to a circle formation. I guess it has to do with some molecular forces, but the details are not clear to me.
BTW: if I understand you correctly, the insert itself (the megaprimer) is never copied/created during the second PCR and thus this is only formed during the first PCR. This makes the amount of starting material (of megaprimer) pretty important.

#6 Re: rf-cloning troubles » Is phosphorilation needed? » 2015-01-17 16:23:35

Steve Bond wrote:

Hi Sand,
You're not alone in misunderstanding how the rf-cloning method leads to a circularized product. Some mental gymnastics are involved.
It all has to do with the positioning of priming sites. Both the 3' and 5' end of a good mega-primer will be bound to it's target parental plasmid, with some non-complementary sequence in the middle. As you know, priming is initiated at the 3' end, and then, because the DNA polymerase does not have 5'-3' exonuclease activity, synthesis stops when the daughter strand bumps into the 5' end of the mega-primer. This happens on the complement strand as well, and two nicks are present when these newly synthesized products anneal — offset by the length of the mega-primer.
In the next round of PCR, if a mega-primer binds to a daughter product, the 3' end it finds itself at the extreme 5' of the template. Meaning there is nothing to synthesize in the 5'-3' direction, and this is why the 2° PCR of an RF-cloning project is non-exponential.
I hope this clarifies, but I can imagine it's still a bit confusing. The best I can do is promise that the logic is sound, and encourage you to draw the process out on paper until you see how the pieces all fit together. You've probably already looked at it, but it might be useful to check out the diagram in the Q & A again.
Take care,
-Steve

But I understand this.
This is not my problem. The problem is that I do not see why in the RF system you end up with "plasmid" DNA (circle) while in the EMP you end up with a linear piece of DNA...

When you denature your DNA to amplify it => you have a linear piece of DNA ... (or am I missing something here..)
I just do not see why during RF the DNA would stay circulated while in the EMP it will not.
The shape/form or where the primers bind, I do not see how this can attribute to staying as a circle form or a linear form.


Are you saying it stays as a circle because you have a non exponential cloning in the 2th PCR ? I do not see what this has to do with the "form" of the DNA?

I understand that the parental part stays as a plasmid (its not nicked) but the generated piece of DNA , by the PCR, is nicked, so this will linearize. So why would this form a plasmid (circle) later?

#7 Re: rf-cloning troubles » Is phosphorilation needed? » 2015-01-17 14:33:28

Steve Bond wrote:

Hey Gabriele,
I think the difference is that the exponential Megaprimer method leads to completely linear vector which needs to be blunt end ligated, while the more standard version of RF-Cloning gives you a circularized, albeit nicked, plasmid. The transformed cells will repair the nicks all on their own, but are just going to degrade the linear DNA; hence the phosphorylation/T4-ligase step. I'm just skimming through the 2014 article by Peleg and Unger again (doi: 10.1007/978-1-62703-764-8_6) and they are not using the exponential method, which is probably why you aren't seeing reference to a ligation step.
Hope this clarifies!
Good luck, and it would be awesome if you share your experience with the exponential method after you've given it a whirl.
-Steve

I find this an interesting question.

But I find it hard to understand why the RF (traditional method) leads to a more plasmid type shaped product while the EMP method leads to "perfect" linearized products.
Why is this?
I do not see why one method keeps the plasmid shape and the other does not. Both are nicked plasmids, so why does the nicked plasmid stays a "plasmid" in the RF method, but not in the EMP method?

#8 Re: rf-cloning troubles » Question on the basics » 2014-08-11 19:49:27

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.

#9 rf-cloning troubles » Question on the basics » 2014-08-11 18:40:16

Sand
Replies: 3

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)

#10 Re: rf-cloning troubles » Removing part » 2014-07-25 19:00:13

beako1980 wrote:

You sure can!
When you use the website to design your project, place the '!' points on either side of the sequence you want removed.
If you want, I can have a look at your project before you order the primers.
-Steve

If I understand you correctly, than the piece of the plasmid between the "!" marks will be removed and replaced by the insert , right?
Or?

eg.: ...TTTTTT ! AAAAAA ! TTTTTTT...

and the insert would be GGGG

than the plasmid would become ....TTTTT  GGGG TTTTTT... ?


It would be nice yes, if you take a look at it. I will get back in touch with the sequence of the plasmid and the gene I would like to introduce.

Simple put: I have a plasmid in which I want to remove a part and "replace" it with another part.
The plasmid however is rather big!

#11 rf-cloning troubles » Removing part » 2014-07-25 18:00:03

Sand
Replies: 3

Dear all,

I am a complete newbie in this technique and I was wondering if it is possible to (at the same time as inserting a new piece) remove a piece from your vector.

My goal is actually to replace a part of my plasmid with another part.

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