The plasmid "miniprep " method is useful for preparing partially purified plasmid DNA in small quantities from a number of transformants. It relies on an alkaline SDS lysis to free the plasmid DNA from the cell, leaving behind the E. coli chromosomal DNA with cell wall debris. The protocol described involves three basic steps: growth of bacteria and amplification of the plasmid; harvesting and lysis of the bacteria; and purification of the plasmid DNA.
These purification procedures exploit in one way or another the two major differences between Escherichia coli DNA and plasmid DNA:
The purification protocol therefore involves a differential precipitation step,
in which the long strands of E. coli DNA, entangled in the remnants of
lysed cells, are preferentially removed. Because each of the complementary
strands of plasmid DNA is a covalently closed circle, the strands cannot be
separated (without breaking one of them) by conditions such as exposure to mild
alkali (up to pH 12.5), which break most of the hydrogen bonds of DNA. Closed
circular molecules regain their native configuration when returned to neutral
pH.E. coli remains in the denatured state.
This method
provides enough
purified plasmid DNA for sequencing.
5 ml LB medium were inoculated
with a single bacterial colony. The tube was incubated at 37psy176 C
overnight with vigorous shaking. 4.5 ml of the culture were centrifuged
for 20 minutes at 3500 rpm at 4psy176 C. The remainder of the overnight
culture was stored at 4psy176 C. The medium was removed, leaving the bacteria
pellet as dry as possible. The pellet was resuspended in 150 
l ice-cold
Lysis buffer I (50 mM glucose, 10 mM EDTA, 25 mM Tris, pH 8.0) and stored for 5
minutes at room temperature. After adding 300 l freshly prepared Lysis
buffer II (0.2 N NaOH, 1% SDS) and mixing by inversion, the mixture was
incubated for 5 minutes on ice. Now 225 l ice-cold 3M KAc/5M HAc (pH 6.0)
were added and mixed gently. The tube was stored on ice for 5 minutes,
precipitating the chromosomal bacteria DNA. After centrifugation for 15 minutes
at 13000 rpm at 4psy176 C, the supernatant was transferred to a fresh tube.
Proteins were removed by vortexing with 400 l phenol (see section
phenol), adding 300 l Sevac and centrifuging for 2 minutes at
13000 rpm.
500-600 l of the aqueous layer were removed and mixed with 1 ml ethanol to
precipitate the DNA (see section EtOH). After incubating for 5 minutes
at room temperature, the Eppendorf tube was centrifuged for 10 minutes at
13000 rpm at
4psy176 C. The supernatant was removed, the pellet washed with 70% ethanol
and recentrifuged. After vacuum drying, 50 l of TE (pH 8.0) were added.
After addition of 1.5 l RNase A (10mg/ml), the mixture was incubated for
20 minutes at room temperature to remove RNA. The positive result of the
miniprep was guaranteed by analytical digest with EcoRI of a 5
l sample.