Download Practical Hints: Lysis of bacterial cells for plasmid purification

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QIAGEN
E X T R A S
001
Lysis of bacterial cells for
plasmid purification
Pr act ica l
Hi nts
Effective lysis of bacterial cells is a key step in plasmid isolation
as DNA yield and quality depend on the quality of cell lysate
used for the purification. This article describes the principle
behind bacterial lysis, in particular alkaline lysis, and gives hints on how to ensure that
lysis proceeds optimally.
Alkaline lysis
Alkaline lysis is one of the most commonly used methods for
1. Resuspension
lysing bacterial cells prior to plasmid purification (1, 2), and
E. coli cell
optimized alkaline lysis is part of all QIAGEN® plasmid
purification protocols. Production of alkaline lysates involves
four basic steps (Figure 1):
2. Lysis
NaOH/SDS
1. Resuspension
Harvested bacterial cells are resuspended in Tris·Cl–EDTA
buffer containing RNase A.
3. Neutralization
KAc
Tip
Ensure that bacteria are resuspended completely
leaving no cell clumps in order to maximize the
number of cells exposed to the lysis reagents.
Tip
Buffer volumes for alkaline lysis in QIAGEN
4. Clearing of
lysates
plasmid purification procedures are optimized for
Centrifugation
Filtration
particular culture volumes in LB medium. Do not use
a culture volume larger than recommended in the
protocol as this will lead to inefficient lysis and
Supernatant
containing
plasmid DNA
reduce the quality of the plasmid preparation.
Tip
KDS precipitate
containing
genomic DNA
+ proteins
For large scale purification of low-copy plasmids,
for which larger cultures volumes are used, it may
be beneficial to increase the lysis buffer volumes in
order to increase the efficiency of alkaline lysis
and thereby the DNA yield (Figure 2).
Filtrate
containing
plasmid DNA
Plasmid DNA
Chromosomal
DNA
Figure 1 Principle of alkaline lysis
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Amount of plasmid DNA in cleared lysate
2. Lysis
High volumes of lysis buffers
Cells are lysed with NaOH/SDS. Sodium
dodecyl sulfate (SDS) solubilizes the
phospholipid and protein components of
the cell membrane, leading to lysis and
Low volumes of lysis buffers
release of the cell contents. NaOH
denatures the chromosomal and plasmid
DNAs, as well as proteins. The presence
of RNase A ensures that liberated cellular
Culture volume
RNA is digested during lysis.
Figure 2 The effect of lysis buffer volumes on the amount of low-copy plasmid DNA in
the cleared lysate for large-scale plasmid preparations.
Tip
Avoid vigorous stirring or vortexing of the lysate as this can shear the bacterial
chromosome, leaving free chromosomal fragments in the supernatant which will
copurify with the plasmid DNA. The solution should be mixed gently but thoroughly by
inverting the lysis vessel 4–6 times.
Tip
Do not allow the lysis to proceed for longer than 5 minutes. A 5-minute incubation
allows maximum release of plasmid DNA from the cell, while minimizing the release
of chromosomal DNA and reducing the exposure of the plasmid to denaturing conditions.
3. Neutralization
The lysate is neutralized by the addition of acidic potassium acetate. The high salt concentration
causes potassium dodecyl sulfate (KDS) to precipitate, and denatured proteins, chromosomal
DNA, and cellular debris are coprecipitated in insoluble salt-detergent complexes. Plasmid
DNA, being circular and covalently closed, renatures correctly and remains in solution.
Tip
Precipitation is enhanced by using chilled neutralization buffer and incubating on ice.
(For many plasmid miniprep procedures, use of chilled buffer and incubation on ice
are not necessary.)
Tip
Mix the solution gently but thoroughly to ensure complete precipitation.
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References
4. Clearing of lysates
1. Birnboim, H.C., and
Doly, J. (1979) A rapid
alkaline lysis procedure
for screening recombinant
plasmid DNA. Nucl. Acids.
Res. 7, 1513–1522.
Precipitated debris is removed by either high-speed centrifugation or filtration, producing
cleared lysates.
Tip
Lysate filtration technologies developed by QIAGEN ensure removal of all precipitated
2. Birnboim, H.C. (1983) A
rapid alkaline extraction
method for the isolation of
plasmid DNA. Methods
Enzymol. 100, 243–255.
material without centrifugation, reducing plasmid purification time considerably,
particularly when large culture volumes or large numbers of samples are involved.
Specialized modules are available for different scales of plasmid preparation, as well
3. Sambrook, J., Fritsch, E.F.,
and Maniatis, T. (1989)
Molecular cloning: A Laboratory manual. Cold
Spring Harbor: Cold
Spring Harbor Laboratory.
as for high-throughput plasmid minipreps.
Other lysis methods
4. Ausubel, F.M., et al.
(1987) Current Protocols
in Molecular Biology.
New York: John Wiley
and Sons.
A number of other methods have been described for lysing bacterial cells (3, 4). Some of these
methods were developed for different applications and may not be suitable for plasmid
DNA isolation.
Boiling lysis: Bacterial cells are treated with lysozyme to weaken the cell walls and then lysed
by heating in a boiling water bath for ~1 minute.
Lysis with detergent: Bacterial cells are lysed by treatment with an ionic detergent (e.g., SDS)
or a nonionic detergent (e.g., Triton® X-100).
Mechanical lysis: Bacterial cells are lysed by mechanical disruption, for example by sonification.
Enzymatic digestion: Some lysis methods include treatment of bacteria with enzymes such as
lysozyme which assist in weakening cell walls.
Lysis of bacteria other than E. coli
Isolation of plasmid DNA from bacteria other than E. coli usually requires modifications to the
lysis procedure in order to optimize lysis conditions for the particular species. Detailed
protocols for plasmid DNA isolation from a variety of bacteria using QIAGEN plasmid
purification procedures are available on request from QIAGEN Technical Services or your local
distributor.
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