Transformation technique's and [abx] affect on plasmid yield - plasmid production and isolation (Dec/19/2001 )
Can anyone tell me whether or not the bacterial transformation technique affects the copy number of a given plasmid? For instance, if I transform DH5alpha cells with a pUC based plasmid via the CaCl2 protocol and the electrocomp protocol, will one 'cell bank' start cultures that will yield more plasmid DNA than cultures started with the other cell bank?
Also, will all DH5alpha transformants on a given plate yield the same amount of plasmid DNA? If not, what factors affect the ultimate yields.
Finally, some references suggest that it is wise to use a higher antibiotic concentration to select for plasmid containing host cells then is necessary. For instance, I have seen one paper that suggests a Kanamycin concentration of 100 mg/L as opposed to 50 mg/L. It seems logical that this would help select for cells with more plasmids but does anyone know if this is actually the case? Perhaps it is more logical to think that an Ampicillin plasmid would work better in this model considering the proteinaceous resistance mechanism.
Thank you very much for your time. Any thoughts on this subject are greatly appreciated.
I have been isolating plasmids from transformants regularly and my yields from different cultures do not differ greatly. Ofcourse not all the bacterial cells will not have exactly the same number of the plasmid, but the difference should not be great.
Increasing the concentration of the antibiotic only helps keep the selection pressure for a longer time especially when cultures are grown overnight. If the concentration is too low the antibiotic would be degraded soon and due to absence of pressure the cells would tend to lose the plasmid too.