Professional Documents
Culture Documents
Lysis of Plasmidian Cell
Lysis of Plasmidian Cell
E X T R A S QIAGEN
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
23
1011204_QNews299_JDist.qxd 25.02.1999 15:23 Uhr Seite 24
QIAGEN E X T R A S
002
2. Lysis
Amount of plasmid DNA in cleared lysate
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
RNA is digested during lysis.
Culture volume
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.
Avoid vigorous stirring or vortexing of the lysate as this can shear the bacterial
Tip 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.
Do not allow the lysis to proceed for longer than 5 minutes. A 5-minute incubation
Tip 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
Mix the solution gently but thoroughly to ensure complete precipitation.
24
1011204_QNews299_JDist.qxd 25.02.1999 15:23 Uhr Seite 25
E X T R A S QIAGEN
003
4. Clearing of lysates References
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.
QIAGEN Practical Hints for daily laboratory practice will continue in future issues of
QIAGEN News. If there is any other information you would like to see on these pages of
QIAGEN News, please let us know by calling QIAGEN Technical services or your local
distributor.
Signature: Date: 25
Witness: Date: