Is it possible to do transformation without using plasmid? Can I directly force DNA fragments from PCR into a bacterium without ligating it into a plasmid? And how do I do it? (I am not using E.coli)
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.
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
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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.