The method you choose depends on the type of experiment you are performing and the desired result.

(1) Are you transforming B. subtilis with chromosomal DNA? Then you use linear fragments. These can either be a simple genomic DNA prep, if you are moving a genetic marker from one strain to another, or can be generated in vitro by PCR. Keep in mind that if you are integrating a marker at a particular site, you need at least 500 bp of chromosomal homology flanking both sides of the marker for efficient transformation. Regardless, for this type of experiment you should use naturally competent cells and linear DNA fragments.

(2) Are you transforming B. subtilis with an integrative vector and is targeted to a particular site on the chromosome, such as the amyE locus? In that case you perform the cloning in E. coli in an appropriate vector. (You can contact me directly for suggestions if you wish.) You isolate the plasmid DNA and linearize it by digesting with a single site restriction enzyme that cuts the plasmid backbone outside the integration cassette. Then you transform naturally competent B. subtilis cells with linear DNA fragments.

(3) Are you transforming B. subtilis with a replicative vector? If so, you are probably using a shuttle vector. In that case, you once again perform the cloning in E. coli and isolate plasmid DNA. At that point you can either transform naturally competent or electrocompetent B. subtilis hosts with the circular plasmid. If you are using natural competence, as most people do, then multimeric forms are required. No problem; the E. coli plasmid prep will have some of them. In either case it will help efficiency significantly to use a restriction-minus host of B. subtilis, such as strain 1012 (BGSC accession 1A982).

There are specialize situations, such as introducing huge chunks of linear DNA, using B. subtilis as a "genome vector," but those three cover most of the kinds of experiments that you are likely to do.

Dear,

First of all, you have to targeted a DNA segment or gene from DNA plasmid or DNA or genomic of Bacillus subtilis, then you digest this DNA by a restriction enzymes. 

Secondly you have to clone this DNA segment into target host cell like DH5alpha cells (strain from e.coli).

https://microbewiki.kenyon.edu/index.php/DH5-Alpha_E.coli

http://www.ncbi.nlm.nih.gov/pubmed/15982188

Kindly write to me for any further information

Thank you

Hi Lizett, both will work. I have transformed B. subtilis with complete plasmids as well as linear (PCR) fragments and it worked in both cases. Important is to get them in a nice competent stage. 

good luck.

The method you choose depends on the type of experiment you are performing and the desired result.

(1) Are you transforming B. subtilis with chromosomal DNA? Then you use linear fragments. These can either be a simple genomic DNA prep, if you are moving a genetic marker from one strain to another, or can be generated in vitro by PCR. Keep in mind that if you are integrating a marker at a particular site, you need at least 500 bp of chromosomal homology flanking both sides of the marker for efficient transformation. Regardless, for this type of experiment you should use naturally competent cells and linear DNA fragments.

(2) Are you transforming B. subtilis with an integrative vector and is targeted to a particular site on the chromosome, such as the amyE locus? In that case you perform the cloning in E. coli in an appropriate vector. (You can contact me directly for suggestions if you wish.) You isolate the plasmid DNA and linearize it by digesting with a single site restriction enzyme that cuts the plasmid backbone outside the integration cassette. Then you transform naturally competent B. subtilis cells with linear DNA fragments.

(3) Are you transforming B. subtilis with a replicative vector? If so, you are probably using a shuttle vector. In that case, you once again perform the cloning in E. coli and isolate plasmid DNA. At that point you can either transform naturally competent or electrocompetent B. subtilis hosts with the circular plasmid. If you are using natural competence, as most people do, then multimeric forms are required. No problem; the E. coli plasmid prep will have some of them. In either case it will help efficiency significantly to use a restriction-minus host of B. subtilis, such as strain 1012 (BGSC accession 1A982).

There are specialize situations, such as introducing huge chunks of linear DNA, using B. subtilis as a "genome vector," but those three cover most of the kinds of experiments that you are likely to do.

Thanks to all of you, Dr. Daniel Ziegler, your answer was very clear and complete, thank you for your help!!!

Daniel Zeigler, which amount of PCR product do you suggest to use to transform Bacillus subtilis?