Elif is right. If the PCR fragment isn't targeted somewhere in the genome for integration, it will be degraded and lost.  

there is an experiment of griffith which discusses transformation where there are chances of uptake of DNA, though there was no point mentioned there that those rough live strains were made competent or any other discussion as such.so there may be a possibility that may happen.

or u can used other than ecoli any other strain which is used to guide the linearized DNA using Cre LoX principle.

hope this would help. 

What about selection marker? How will you select colonies? How would it replicate in the host without the origin of replication (ORI)? 

Hi Daniel,

as mentioned above, bacteria will only replicate circular DNA that contains an appropriate Ori sequence. However, most bacteria strains are able to repair DNA that is not circular but anneals to itself. If you have DNA from PIPE cloning or similar methods, then you can transform without prior ligation, since the bacteria are able to "repair" the self-annealed vector into a functional circular molecule. Beware of frameshifts, though, when using this method.

DNA fragments can enter, but what is your purpose? Are you trying to integrate foreign DNA into the bacterial genome or just to make bacteria producing your protein of interest? In the second case, plasmid cloning is very simple and effective. If you are trying to reach a different goal, you can explore alternative methods already described.

For genetic transformation, people have successfully transformed following materials/molecules into cells:  (1) protein, (2) RNA , (3) plasmid DNA, (4) linear DNA, (5) short synthetic oligo DNA fragment. It depends on your research goal, experimental organism to use them. Different methods can be used to deliver, ex. electroporation, bombardment (gene gun), PEG-mediated (ex. plant protoplast transformation), microinjection (mammalian cells), other material mediated mammalian cell transformation.......

Hello everyone. I am trying to do a gene knock out in Burkholderia ubonensis by inserting a Kanamycin resistance gene in the target gene. I was wondering if homologous recombination still take place if I dont put the DNA fragment from PCR into a plasmid. The method I am using right now is time consuming. After PCR, I ligate DNA fragment into pGemTeasy vector--->transformation----> select the mutants through PCR----> plasmid extraction---> Ecor1 Digestion---> ligate the digested DNA into another plasmid---> transformation again---> select the mutants---> extract the plasmid---> electroporate the plasmid into ubonensis and check for the phenotype. I was wonder if i can skip all of these steps by directly electroporate my DNA fragment from PCR into ubonensis

PS: Is there a way to reply to individual answer?

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Yes, you can directly transfer your PCR Product into bacteria through electroporation (see attached paper, yellow highlights). Once the PCR products are inside the bacterial cells, you need some kind of help for homologous recombination (HR) to occur or to have higher HR efficiency. They call this whole process: "Recombineering". In the attached paper, a bacterial strain expressing a bacteriophage recombination system was used. The protein(s) from the phage recombination system promote HR between the PCR product and genomic DNA.

I am not sure whether the HR frequency (between PCR product and genomic DNA) will be very low or HR would not occur at all if no such 'phage recombination system' present.

yes of course you can do this. The original Avery experiments showing DNA was the genetic material was precisely this type of experiment. HOWEVER it very much depends upon the bacteria that you use. For example, you will essentially never get transformation with a PCR product in E. coli unless you remove certain specific proteins in the recombination system (either by deletion or by expressing an inhibitor protein, this is the background for doing recombineering). In other bacteria, transformation with linear genomic DNA works fine. It comes down to efficiency of transformation and the nature of the recombination enzymes in the bacterial host. So there is no simple answer, it depends upon the bacterium.