Experiment 3 for CSS451

Plasmid Isolation Using Alkaline Lysis

Plant Biotechnology Resource & Outreach Center, Michigan State University

Plasmid Isolation Protocol

1. 5 ml LB medium containing proper antibiotics were inoculated with a single

bacterial colony. The tube was incubated at 37 C overnight with vigorous
shaking at 360 rpm.
2. Pellet bacteria from the culture at 10,000 x g for 5 minutes at room temperature.
3. Discard the supernatant.
4. Resuspend bacterial pellet in a total of 1 ml ice-cooled solution I (50 mM). Pipet
up and down or vortex as necessary to fully resuspend the bacteria.
5. Add 2 ml room temperature 0.2 N NaOH/1.0% SDS to the suspension. Mix
thoroughly by repeated gentle inversion. Do not vortex.
6. Add 1.5 ml ice-cold Solution III to the lysate. Mix thoroughly by repeated gentle
inversion. Do not vortex.
7. Centrifuge at 15,500 x g for 30 minutes at 4C.
8. Recover resulting supernatant.
9. Add 2.5 volume isopropanol to precipitate the plasmid DNA. Mix thoroughly by
repeated gentle inversion. Do not vortex.
10. Centrifuge at 15,500 x g for 30 minutes at 4C.
11. Removal of resulting supernatant. The pellet is plasmid DNA.
12. Rinse the pellet in ice-cold 70% EtOH and air-dry for about 10 minutes to allow
the EtOH to evaporate.
13. Add ddH2O or TE to dissolve the pellet. After addition of 2ul RNase A
(10mg/ml), the mixture was incubated for 20 minutes at room temperature to
remove RNA.

Note:

1. Spin down your cells. Your DNA is still in the cells, so it is in the pellet at this
stage.

2. Discard the supernatant and to even invert the tube and wipe the lip with a
Kim-wipe or Q-tip.

3. Resuspend the cells in buffer (often Tris) and EDTA. EDTA chelates divalent
metals (primarily magnesium and calcium). Removal of these cations destabilizes
the cell membrane. It also inhibits DNases. Glucose should also be added to
maintain osmolarity and prevent the buffer from bursting the cells.

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 4. Lyse the cells with sodium hydroxide (NaOH) and SDS. This highly alkaline
solution gave rise to the name of this technique. Mix this by gentle inversion and
incubate on ice for five minutes (but no longer, or your DNA will be irreversibly
denatured). Three things happen during this stage:

a. SDS pops holes in the cell membranes. SDS (sodium dodecyl (lauryl) sulfate)
is a detergent found in many common items such as soap, shampoo and toothpaste.

b. NaOH loosens the cell walls and releases the plasmid DNA and sheared
cellular DNA.

c. NaOH denatures the DNA. Cellular DNA becomes linearized and the strands
are separated. Plasmid DNA is circular and remains topologically constrained.

5. Renature the plasmid DNA and get rid of the garbage. Add potassium
acetate (KAc), which does three things:

a. Circular DNA is allowed to renature. Sheared cellular DNA remains denatured

as single stranded DNA (ssDNA).

b. The ssDNA is precipitated, since large ssDNA molecules are insoluble in high
salt.

c. Adding sodium acetate to the SDS forms KDS, which is insoluble. This will
allow for the easy removal of the SDS from your plasmid DNA.

Now that you've made it easy to separate many of the contaminants, centrifuge to remove
cell debris, KDS and cellular ssDNA. Your plasmid DNA is in the supernatant, while all
of the garbage is in the pellet.

6. Precipitate the plasmid DNA by alcohol precipitation (ethanol or

isopropanol) and a salt (such as ammonium acetate, lithium chloride, sodium
chloride or sodium acetate) and spin this down. DNA is negatively charged, so
adding a salt masks the charges and allows DNA to precipitate. This will place
your DNA in the pellet.

7. Rinse the pelletyour plasmid DNAin ice-cold 70% EtOH and air-dry for
about 10 minutes to allow the EtOH to evaporate.

8. Resuspend your now clean DNA pellet in buffer (often Tris) and EDTA plus
RNases to cleave any remaining RNA. Your DNA is now back in solution.

DNA of this purity is good for a number of uses, such as in vitro transcription or
translation or cutting with some enzymes. If you are sequencing or transforming this
DNA into mammalian cells, you'll want to use additional purification techniques such as
phenol extraction, Qiagen column purification, or silica-based purification.

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Isolation of the Plasmid after Alkaline Lysis

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:

1. The E. coli chromosome is much larger than the DNA of plasmids used as vectors.
2. The bulk of E. coli DNA extracted from cells is obtained as broken, linear
molecules. By contrast, most plasmid DNA is extracted in a covalently closed,
circular form.

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 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

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supernatant was removed, the pellet washed with 70% ethanol and recentrifuged. After
vacuum drying,

Solution I (Lysis buffer I): 50 mM glucose, 10 mM EDTA, 25 mM Tris, pH 8.0. Store

at 0C

a. 10ml 500mM Glucose

b. 2ml 500mM EDTA pH 8.0
c. 2.5ml 1M Tris pH 8.0
d. 85.5ml H2O

e. Autoclave and store at 4C

Solution II (Lysis buffer II): Freshly prepared 0.2 N NaOH, 1% SDS. Store at room
temperature (RT)

Isopropanol: Stored at -20 0C

Solution III (Lysis buffer III): 3M KOAc, pH 6.0

a. 60ml 5M potassium acetate (49.07g potassium acetate in 100ml H2O)

b. 11.5ml glacial acetate
c. 28.5ml H2O