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Electroporation and marker exchange of Pseudomonas syringae pv. syringae.
Laboratory Exercise I: Introduction of broad host range plasmids into P. syringae pv. syringae strain B301D.
Laboratory Exercise II: Introduction of a broad host range plasmid into various Pseudomonas syringae pv. syringae strains.
Laboratory Exercise III: Introduction of mutated genes into Pseudomonas syringae pv. syringae strain B301D, selection for marker exchange, and screening strains for phenotypic changes.
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Electroporation and marker exchange of Pseudomonas syringae pv. syringae.
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Section III, Note 4:
Section III, Note 4:
During electroporation, there is a possibility of the sample "arcing". This is characterized by a loud "pop" sound. Arcing occurs when the sample is too conductive. There are several reasons that a sample will arc including: not washing all of the salt from the growth medium out of the bacterial suspension; placing too much DNA in the electroporation mix; using DNA dissolved in a high salt buffer; using a bacterial suspension that is too concentrated; using a bacterial suspension containing lysed bacteria; and trying to electroporate using electroporation cuvettes that are too warm. If arcing occurs, dilute the DNA and try again, this is usually the most common problem. If arcing continues, perform an electroporation without DNA to see if the cells are too concentrated. Expect a time constant over 4 seconds; electroporation at time constants below 4 seconds will not produce good efficiencies of transformation and should be repeated with less DNA.