Anything and everything cloning: Go...
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I think I get what you're saying, and it may work... I worry that you're less likely to get the exact sequence you want though at the end of the day, because DS break repair/recombination can be a messy process. I'd say it might be a fun thing to play around with just to see what happens.
Give it a try
Fascinating discussion!
Check the link to the paper I included (its a free paper).
I am guessing this is what Sand means! I have not fully studied it, but it seems interesting.
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?
Is this, perhaps not what you mean: http://www.sciencedirect.com/science/ar … 6114200161
I am not sure I understand you and the method (I just glanced at it, I was looking for further information because I want to try the RF-cloning in the near future.
So I stumbled upon this (above mentioned) paper!
Hey Jan,
The amount of megaprimer to use is provided in the output as 'insert', and is expressed as 'ng' instead of a μM concentration.
-Steve
Aha!
Yes I was looking here: http://www.rf-cloning.org/QandA.php, but indeed if you use the website to make the primers (where you put the plasmids) it tells you have much you need! haha , silly me.
Another question, not so much related to the restriction free cloning itself: what kind of kit do you use to do the gel extraction. THe kit I am using (for a different project) has low yields of DNA, too low.
Any suggestion?
Dear all,
whats the amount of mega primers you use for the second PCR?
Also 10μM ?
Plasmid template?
I plate the entire 100uL of competent DH5a on ampicillin containing agar plates.
What other sort of cells would you consider? I don't have access many bacterial strains as we always use DH5a in my lab. I also have access to XL-Gold and Top10 cells. If you have any specific strains in mind I can ask around the Microbiology department and see if they have anything I can borrow
I am not an expert on RF cloning, but did you check your competent cells themselves? Transformed them with something you know that should work? Just to see the cells themselves are ok?
Sometimes this is a problem.
Top10 cells , you can try this too.
Another approach: electroporation rather than chemical transformation. Electroporation has a higher efficiency
Hi again Jan,
Sorry for not replying right away, I didn't see your new message until just now.
If you go with the recommended reaction conditions for your particular polymerase, you'll probably be okay, BUT! On your secondary PCR, cut the number of cycles down to 16-18. More cycles will reduce efficiency further.
-Steve
I am going to try it.
I'll use Q5 polymerase, it is supposed to be even better than phusion or is Q5 not the best idea?
What about the reaction settings?
First I'll need to figure out how it works in detail, but I am pretty much going to use it for a plasmid of about 9000bps and 41% GC
The goal is to introduce GFP in the plasmid.
I'll check the website and the tool and if I run into troubles, I'll come back here to ask for more questions.
Hey Jan,
It's a great question. The larger the plasmid, the lower the final efficiency of the reaction, but 10K is not unreasonable. I've personally used RF-cloning for 13K plasmids. The important thing is that you use a polymerase with very high processivity; in other words, an enzyme which does not fall off the guide strand easily. It's the reason I recommend using iProof and Phusion (they are different brand names of the same enzyme), because they have an extra DNA binding domain that gives them a big boost in processivity.
Unless your plasmid is obnoxiously large, I would say 'give it a try'.
Take care,
-Steve
I see.
And you used some sort of specific protocol?
Or just a common PCR protocol for long templates?
Dear all,
a rookie question: to me it seems that one of the pitfalls of this technique is the fact that you use a PCR to generate the novel plasmid (with the insert).
Now I am wondering: what is the max size if a plasmid that can be used?
I can imagine it being very hard to use this techniques with plasmids ranging between 6000bps and 10.000 bps (or bigger)? Or is this not a problem?
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