I had transformed B. subtilis and E. coli cells with same plasmid and transformed cells were allowed to grow on selection medium. Cells grown on selective medium were then subjected to plasmid isolation. Recombinant plasmid was successfully isolated from E. coli but with the same protocol B. subtilis plasmid was not isolated. Kinldly help me to isolate the plasmid from Bacillus cells.
I had good experience with the attached protocol. It is based on the Qiagen miniprep kit and adds some lysozyme to the reaction. Good luck!
If harsh condition was needed for plasmid extraction from Gram +ve bacteria, the bead extraction method by Coton and Coton (experimental section 2.4 in the paper) should contribute to that function, then follow the plasmid purification method you use previously.
Hope this least costly method work well for you.
Link to paper :
https://www.researchgate.net/publication/276929810_Isolation_and_Taxonomic_Identity_of_Bacteriocin-Producing_Lactic_Acid_Bacteria_from_Retail_Foods_and_Animal_Sources
Article Isolation and Taxonomic Identity of Bacteriocin-Producing La...
Hello Suresh,
After few trials and errors, I got successful results with Qiagen plasmid Midi kit. Size of my recombinant plasmid was approx. 10 kb (low copy number) and I remember doing vigorous vortexing after adding the first buffer. All the best.
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