Preparation of Plasmid DNA by Alkaline Lysis with SDS: Mini Preparation
E.coli
Plasmid DNA may be isolated from small scale (1-2 ml) bacterial cultures by treatment with alkaline and SDS. The resulting DNA
preparation may be screened by electrophoresis or restriction digests. With further purification with PEG, the preparations may be used
as templates for DNA sequenceing.
Materials
Alkaline Lysis Solution 1
50 mM glucose
25 mM Tris-Cl (pH 8)
10 mM EDTA (pH 8)
Prepare in 100 ml batches from standard stocks. Autoclave 15 minutes at 15 PSI. Store at 4 °C.
Alkaline Lysis Solution 2
0.2 N NaOH (freshly diluted from a 10 N stock)
1% (w/v) SDS
Prepare Solution 2 fresh for each use. Use at room temperature.
Alkaline Lysis Solution 3
5M Potassium Acetate
(60 ml)
Glacial Acetic Acid
(11.5 ml)
H2O
(28.5 ml)
Store at 4°C and use ice cold.
Suitable antibiotic
Ethanol
Phenol:Chloroform (1:1, v/v)
STET
10 mM Tris-Cl (pH 8)
0.1 M NaCl
1 mM EDTA (pH 8)
5% (v/v) Triton X-100
Verify pH of finished solution is 8.0 before use. Do not sterilize.
TE (pH 8) containing 20 g/ml RNase A
LB media
Methods
Cell Preparation
Incubate 2 ml of LB media containing antibiotic with a single colony of transformed bacteria. Incubate overnight at
37°C with shaking.
Culture tube volume should be at least four times that of the culture, loosely capped and agitated vigorously.
Pour 1.5 ml of culture into a microcentrifuge tube. Spin at top speed for 30 seconds at 4°C. Store remaining culture
at 4°C.
Aspirate supernatant.
Cell Lysis
Resuspend bacterial pellet in 100 L of ice cold Lysis Solution 1 with vigorous vortexing.
Make sure pellet is completely suspended.
Add 200 L of Lysis Solution 2. Invert tubes five times to mix. Do not vortex.. Keep tubes on ice.
Add 150 L ice cold Lysis Solution 3. Invert tube several times to mix. Store on ice for 3-5 minutes.
Spin at top speed for 5 minutes at 4°C. Transfer supernatant to a clean tube.
(Optional) Add an equal volume of phenol:chloroform. Mix by vortexing then spin at top speed for 2 minutes at 4°C.
Transfer the upper aqueous layer to a clean tube.
Plasmid DNA Recovery
Ethanol precipitate the solution (2 volumes of ethanol plus 1/10 th volume of 3M sodium acetate, vortex, let stand 2
minutes).
Spin at top speed for 4 minutes at 4°C to pellet DNA.
Aspirate supernatant completely.
Wash pellet with 1 ml 70% ethanol. Spin at top speed for 2 minutes at 4°C
Aspirate supernatant completely, carefully avoiding pellet.
Allow tube to sit open at room temperature for 5-10 minutes to dry contents.
Dissolve pellet in ~50 L of TE buffer containing 20 ug/ml DNase-free RNase-A. Vortex briefly. Store DNA at 20°C.