WEEK 5 & 6: LAB BIOS390 ERIC KYLE
BIOS .
WEEK 6: LAB: isolate Plasmid DNA
Once a plasmid has been introduced into competent bacterial cells and the cells have grown into
colonies on a medium selective for cells containing the plasmid, the next step is to perform a
miniprep of the plasmid DNA in preparation for DNA sequencing. The three steps are: 1)
growing cells in liquid culture; 2) purifying the plasmid DNA from the culture; and 3)
performing a restriction digest on the purified DNA to determine whether the DNA insert in the
plasmid is the expected size. To start the liquid culture, cells from an isolated bacterial colony
are placed in selective medium (nutrient broth with an antibiotic to which the plasmid provides
resistance). This placing of the cells into the medium is called inoculation. It is important to
choose a single isolated colony from the plate, so that the liquid culture will contain cells that all
have the same plasmid. If cells from more than one colony are used for inoculation, the miniprep
may contain multiple plasmids and the mixed DNA will not be useful for sequencing. The cells
that have grown overnight are harvested by centrifugation and the plasmid DNA is purified. The
purification is a multistep process designed to separate plasmid DNA from genomic DNA
(gDNA) and other cellular components.
BIOS .
WEEK 6: LAB: isolate Plasmid DNA
Once a plasmid has been introduced into competent bacterial cells and the cells have grown into
colonies on a medium selective for cells containing the plasmid, the next step is to perform a
miniprep of the plasmid DNA in preparation for DNA sequencing. The three steps are: 1)
growing cells in liquid culture; 2) purifying the plasmid DNA from the culture; and 3)
performing a restriction digest on the purified DNA to determine whether the DNA insert in the
plasmid is the expected size. To start the liquid culture, cells from an isolated bacterial colony
are placed in selective medium (nutrient broth with an antibiotic to which the plasmid provides
resistance). This placing of the cells into the medium is called inoculation. It is important to
choose a single isolated colony from the plate, so that the liquid culture will contain cells that all
have the same plasmid. If cells from more than one colony are used for inoculation, the miniprep
may contain multiple plasmids and the mixed DNA will not be useful for sequencing. The cells
that have grown overnight are harvested by centrifugation and the plasmid DNA is purified. The
purification is a multistep process designed to separate plasmid DNA from genomic DNA
(gDNA) and other cellular components.
