RF-Cloning.org Forum

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

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?