This week you will isolate plasmid DNA from two colonies that we suspect may carry the
We suspect they may be the desired clones because they were resistant to
chloramphenicol and they were white on X-gal - in other words, based on their phenotype.
week we will begin the analysis of the genotype of these clones - to see if they actually carry the
correct DNA fragments.
We will purify the plasmid DNA from your putative clones, and then
cleave the DNA with restriction endonucleases.
Next week, we will visualize the resulting DNA
fragments using an agarose gel, and thereby determine whether the DNA pieces are the sizes we
would predict for the desired clone.
Small scale plasmid preparation - Miniprep:
(Step numbers correspond to the attached miniprep procedure)
See also figure at the end of this discussion.
Step 1 - Growth of the bacterial culture
- In order to purify enough plasmid DNA to analyze,
we need to begin with more cells than are present in a single colony.
To grow the cells for a
miniprep, we generally use about 5 ml of a liquid culture of the plasmid containing cells.
cells are grown in a rich broth (LB) containing the antibiotic whose resistance is encoded on the
plasmid (in our case Ampicillin and/or Chloramphenicol).
A rich broth is used so that the cells
grow up to a high cell density.
The antibiotic is necessary to prevent loss of the plasmid from the
cells in the culture.
The presence of the plasmid in the cell is something of a burden, so plasmid-
containing cells grow somewhat slower than plasmid-free cells.
If the cells were grown without
the antibiotic, any cells that lost the plasmid (by random chance) would grow faster than the
By the time the cells were harvested, the plasmid-free cells could be a
majority of the population.
The presence of the antibiotic in the medium prevents the growth of
plasmid-free cells, so the cells harvested will almost all carry the plasmid.
Steps 2-4 - Harvesting the bacterial cells -
We want to isolate the plasmid DNA from ~4.5 ml
of culture, but the most convenient sized tubes in which to perform the procedure hold only 1.5
To get around this, we will simply collect the cells from 1.5 ml, pour off the supernatant
(leaving the pelleted cells behind), and then add another 1.5 ml of culture, collect the cells again,
and repeat one more time.
Steps 5-10 - Lyse the cells -
The combination of these steps will break open the cells, releasing
the plasmid DNA.
In step 5, Solution I is added.
Solution I contains a buffer (Tris) to maintain a
constant pH, glucose, and EDTA.
The EDTA chelates (binds tightly to) divalent cations that are
necessary to stabilize the cell membranes.
Addition of EDTA destabilizes the membranes,
making the cells easier to lyse.
In step 8, the cells are treated with Solution II, which contains
both Sodium dodecyl sulfate (SDS - an ionic detergent) and Sodium hydroxide (a strong base).
The SDS dissolves the lipid bilayers of the cell’s membranes and thereby lyses the cells.