RF-Cloning.org Forum

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.

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

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

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

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