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Performed a bacterial conjugation and restriction digest on R. eutropha
to confirm plasmid's presence
Advanced Molecular Biology Project
Bacterial Conjugation
Bacterial Conjugation
E. Coli were grown with phaZ2 plasmid, the recipient R. Eutropha was not grown with a plasmid. A spectrophotometer was used to find the optical density value to determine the final culture volume.
A LB plate was prepared with a filter, the bacterial samples were prepared and allowed to grow on the plate for a week.
Selecting for Transconjugates
Selecting for Transconjugates
Two sterile dilutions were performed on the colonies with an antibiotic to select for the conjugated bacteria.
The dilutions were 1:10 and 1:100, which produced a lawn and a bountiful amount of colonies respectively
Plasmid Harvesting
Plasmid Harvesting
DNA Collection
DNA Collection
Using the Qubit Assay kit, R. Eutropha cultures were suspended and lysed.
Using Buffers, Proteinase K, and Spin columns the genomic DNA of the bacteria had been harvested.
Using the Qubit Assay Fluorometer, the concentration of DNA could be found in the sample, this was to help determine the amount of primer that would be needed.
Restriction Digest
Restriction Digest
The plasmid map was analyzed to determine which restriction digest enzymes would be used.
Three samples of the plasmid were prepared to with different combinations of restriction enzymes.
The the length of the plasmid expected was determined by the plasmid map.
Gel Results
Gel Results
One of the samples was prepared and run on the agarose gel.
The results confirmed the plasmid's identity and matched the expected base pair length indicated by the plasmid map.
Skills Advanced
Skills Advanced
Lab Safety
Gel Loading
Restriction Digest
Plating Technique
Lab Notetaking
Lab Reports
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