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This procedure is used to extract plasmid DNA from bacterial cell suspensions
and is based on the alkaline lysis procedure developed by Birnboim and
Doly (Nucleic Acids Research 7:1513, 1979). The procedure takes advantage
of the fact that plasmids are relatively small supercoiled DNA molecules
and bacterial chromosomal DNA is much larger and less supercoiled. This
difference in topology allows for selective precipitation of the chromosomal
DNA and cellular proteins from plasmids and RNA molecules. The cells are
lysed under alkaline conditions, which denatures both nucleic acids and
proteins, and when the solution is neutralized by the addition of Potassium
Acetate, chromosomal DNA and proteins precipitate because it is impossible
for them to renature correctly (they are so large). Plasmids renature correctly
and stay in solution, effectively separating them from chromosomal DNA
and proteins. Cool, no?
Note: The procedure below is used to make duplicate minipreps. This
provides balanced tubes for the centrifuge as well as twice as much product
when you are finished.
1. Gently swirl the contents of the culture tube to resuspend the cells.
2. Label two 1.5 mL tubes and pipet 1000 uL of the cell suspension into
3. Close the caps and place the tubes in a centrifuge (remember to balance
the centrifuge by putting the tubes opposite one another) and spin at maximum
speed for 20 s.
This part of the procedure collects the bacterial
cells which are suspended in the liquid medium into a cell pellet at the
bottom of the tube.
4. Withdraw and discard the supernatant using a pipettor, being careful
not to disturb the cell pellet. Discard the supernatant in a waste container.
5. Add 100 uL of Buffer 1 (50 mM Tris-HCl, 10 mM EDTA, 100 ug/mL RNase
A, pH 8.0 ) to each tube and resuspend the cells by vortexing. It's very
important that the cell suspension is homogenous and no clumps are visible.
The cells are now resuspended in a buffered solution
with RNase. When the cells are lysed in the next step, the RNase
will catalyze hydrolysis of all RNA molecules into nucleotides, but the
DNA will not be affected.
6. Add 200 uL of Buffer 2 (1% SDS, 0.2 M NaOH ) to each tube. Close the
caps and mix the solutions by rapidly inverting them a few times. DO NOT
VORTEX since the chromosomal DNA released from the broken cells could be
sheared into small fragments and contaminate your plasmid prep.
SDS is an acronym for Sodium Dodecyl Sulfate.
It is an ionic detergent which disrupts cell membranes and destabilizes
all hydrophobic interactions holding various macromolecules in their native
conformation. The high pH of the 0.2 M NaOH also denatures macromolecules
by changing the condition of ionizable groups (ionizing certain groups
and deionizing others). The clearing you see is because the cells
are lysing. The viscosity of the solution is increased by the increase
in concentration of macromolecules in solution (a result of the cell lysis).
7. Let tubes stand on ice for 5 minutes (it's OK if they go longer).
8. Add 150 uL of ice-cold Buffer 3 (3.0 M Potassium Acetate, pH 5.5
) to each tube. Close the caps and mix the solutions by rapidly inverting
them a few times. A white precipitate will form.
This is really the key step in the alkaline lysis
procedure. The low pH of the Potassium acetate solution neutralizes
the NaOH and when the pH returns to near-neutrality then the macromolules
renature. The proteins and large DNA molecules do not renature correctly
however. They form hydrophobic, ionic and hydrogen bonds with each
other nonspecifically because the correct conformation of the molecule
was not maintained during denaturation. The plasmid DNA molecules,
however, never really fully denatured because they are small circular molecules
which are supercoiled. Even though the hydrogen bonds between base
pairs were broken by the high pH, they reform correctly when the pH is
lowered. The large DNA molecules (chromosomal DNA) and proteins form
precipitates because they bind to each other in a large aggregate but the
plasmids don't precipitate because they renature correctly and don't become
part of the large multi-molecule aggregates. Thus plasmid DNA remains
in solution while proteins and other DNA molecules precipitate.
9. Let tubes stand on ice for 5 minutes (it's OK if they go longer).
10. Place the tubes in a centrifuge (balanced) and spin at maximum speed
for 5 minutes. Team up with some other folks on this spin. The precipitate
will pellet along the side of the tube.
11. Transfer the supernatants into clean 1.5 mL tubes, being careful
not to pick up any of the precipitate. Discard the tubes with the precipitate
and KEEP the tubes with the supernatant.
12. Before you do this step, make sure there is a centrifuge available.
To each tube of supernatant add an equal volume (about 400 uL) of isopropanol
to precipitate the nucleic acids. Close the caps and mix vigorously. Let
the tubes stand at room temperature for 2 minutes, place them, with their
hinges pointing outward from the center, in a centrifuge (balanced) and
spin at maximum speed for 5 minutes. This step pellets the nucleic acids
but if you leave it around too long, proteins remaining in solution will
begin to precipitate as well.
This concentration of isopropanol will cause
the DNA to become insoluble. Since DNA molecules are ionic (uniform
negative charge due to the phosphates) they are highly soluble in water
but not soluble in organic solvents. Isopropanol and ethanol are
commonly used to precipitate DNA from aqueous solution.
13. The reason the you oriented the tubes with the hinges outward is because
the plasmid DNA pellet may be difficult to see. It will be at the bottom
and along the hinge side of the tube. Carefully remove and discard the
supernatant. The pellet is usually visible at this point. If not, do not
despair. It may be too small to see but there is probably enough DNA there.
Remember that we are dealing with small molecules that we can't see unless
there is a whole lot of them together!
14. Add 200 uL of absolute ethanol to each tube and mix by inversion
15. Spin the tubes at maximum speed in a centrifuge for 2-3 minutes
This "ethanol wash" will speed up the process
because the next step is to evaporate the alcohol used in the precipitation
step. Absolute Ethanol evaporates much faster than a solution of
16. Carefully remove and discard the supernatant. Try to get as much out
as possible without dislodging the pellet of plasmid DNA.
17. Place the tubes in the fume hood with the caps open for 15-20 minutes
to dry off the last traces of ethanol.
18. When the ethanol is gone (you can check this by smelling the tube)
add 20 uL of TE buffer (10 mM Tris-HCl, 1 mM EDTA, pH 8.0) to dissolve
the pellet. Pipet the 20 uL in and out, up the side of the tube to ensure
that all of the plasmid DNA comes into contact with the TE buffer.
TE buffer is commonly used to redissolve DNA
because it contains EDTA. The EDTA will chelate magnesium ions which
are a cofactor for most nucleases (enzymes which degrade nucleic acids).
If your DNA prep becomes contaminated with a nuclease (like the ones produced
by the cells in your skin) then the nuclease will be inactivated by the
fact that the magnesium cofactor is unavailable in the solution (because
it is chelated by the EDTA).
19. Pool the two 20 uL solutions into one labeled tube and store
it in the freezer.
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Sep 7, 2000 Copyright (C) 1996, Ivor
Knight and Jonathan Monroe. All