Preparation of Plasmid DNA by Alkaline Lysis with SDS: Minipreparation

Plasmid DNA is isolated from small-scale (1-2 ml) bacterial cultures by treatment with alkali
and SDS.

MATERIALS
Buffers and Solutions
Alkaline lysis solution I
Alkaline lysis solution II
Alkaline lysis solution III
Antibiotic for plasmid selection
Ethanol
Phenol:chloroform (1:1, v/v)
STE
TE (pH 8.0) containing 20 g/ml RNase A
Media
Rich medium

METHOD
1. Inoculate 2 ml of rich medium (LB, YT, or Terrific Broth) containing the appropriate antibiotic
with a single colony of transformed bacteria. Incubate the culture overnight at 37C with
vigorous shaking.

2. Pour 1.5 ml of the culture into a microfuge tube. Centrifuge at maximum speed for 30
seconds at 4C in a microfuge. Store the unused portion of the original culture at 4C.

3. Remove the medium by aspiration, leaving the bacterial pellet as dry as possible.
4. Resuspend the bacterial pellet in 100 l of ice-cold Alkaline lysis solution I by vigorous
vortexing.

5. Add 200 l of freshly prepared Alkaline lysis solution II to each bacterial suspension. Close
the tube tightly, and mix the contents by inverting the tube rapidly five times. Do not vortex!
Store the tube on ice.

6. Add 150 l of ice-cold Alkaline lysis solution III. Close the tube and disperse Alkaline lysis
solution III through the viscous bacterial lysate by inverting the tube several times. Store the
tube on ice for 3-5 minutes.

7. Centrifuge the bacterial lysate at maximum speed for 5 minutes at 4C in a microfuge.

Transfer the supernatant to a fresh tube.

8. (Optional) Add an equal volume of phenol:chloroform. Mix the organic and aqueous phases
by vortexing and then centrifuge the emulsion at maximum speed for 2 minutes at 4C in a
microfuge. Transfer the aqueous upper layer to a fresh tube.

9. Precipitate nucleic acids from the supernatant by adding 2 volumes of ethanol at room
temperature. Mix the solution by vortexing and then allow the mixture to stand for 2 minutes at
room temperature.

10. Collect the precipitated nucleic acids by centrifugation at maximum speed for 5 minutes at
4C in a microfuge.

11. Remove the supernatant by gentle aspiration as described in Step 3 above. Stand the tube
in na inverted position on a paper towel to allow all of the fluid to drain away. Use a Kimwipe or
disposable pipette tip to remove any drops of fluid adhering to the walls of the tube.

12. Add 1 ml of 70% ethanol to the pellet and invert the closed tube several times. Recover the
DNA by centrifugation at maximum speed for 2 minutes at 4C in a microfuge.

13. Remove all of the supernatant by gentle aspiration as described in Step 3.Take care with
this step, as the pellet sometimes does not adhere tightly to the tube.

14. Remove any beads of ethanol that form on the sides of the tube. Store the open tube at
room temperature until the ethanol has evaporated and no fluid is visible in the tube (5-10
minutes).

15. Dissolve the nucleic acids in 50 l of TE (pH 8.0) containing 20 g/ml DNase-free RNase A
(pancreatic RNase). Vortex the solution gently for a few seconds. Store the DNA solution at 20C.

RECIPES
Alkaline Lysis Solution I
50 mM glucose
25 mM Tris-Cl (pH 8.0)
10 mM EDTA (pH 8.0)
Prepare Solution I from standard stocks in batches of approx. 100 ml, autoclave for 15 minutes
at 15 psi (1.05 kg/cm2) on liquid cycle, and store at 4C.
For plasmid preparation.
Alkaline Lysis Solution II
0.2 N NaOH (freshly diluted from a 10 N stock)
1% (w/v) SDS
Prepare Solution II fresh and use at room temperature.
For plasmid preparation.
Alkaline Lysis Solution III

5 M potassium acetate, 60.0 ml

glacial acetic acid, 11.5 ml
H2O, 28.5 ml
The resulting solution is 3 M with respect to potassium and 5 M with respect to acetate. Store
the solution at 4C and transfer it to an ice bucket just before use.
For plasmid preparation.
EDTA
To prepare EDTA at 0.5 M (pH 8.0): Add 186.1 g of disodium EDTA2H2O to 800 ml of H2O.
Stir vigorously on a magnetic stirrer. Adjust the pH to 8.0 with NaOH (approx. 20 g of NaOH
pellets). Dispense into aliquots and sterilize by autoclaving. The disodium salt of EDTA will not
go into solution until the pH of the solution is adjusted to approx. 8.0 by the addition of NaOH.
Glycerol
To prepare a 10% (v/v) solution: Dilute 1 volume of molecular-biologygrade glycerol in 9
volumes of sterile pure H2O. Sterilize the solution by passing it through a prerinsed 0.22-m
filter. Store in 200-ml aliquots at 4C.
LB
deionized H2O, to 950 ml
tryptone, 10 g
yeast extract, 5 g
NaCl, 10 g
For solid medium, please see Media Containing Agar or Agarose.
To prepare LB (Luria-Bertani medium), shake until the solutes have dissolved. Adjust the pH to
7.0 with 5 N NaOH (approx. 0.2 ml). Adjust the volume of the solution to 1 liter with deionized
H2O. Sterilize by autoclaving for 20 minutes at 15 psi (1.05 kg/cm 2) on liquid cycle.
Media Containing Agar or Agarose
Prepare liquid media according to the recipes given. Just before autoclaving, add one of the
following:
Bacto Agar (for plates) 15 g/liter
Bacto Agar (for top agar) 7 g/liter
agarose (for plates) 15 g/liter
agarose (for top agarose) 7 g/liter
Sterilize by autoclaving for 20 minutes at 15 psi (1.05 kg/cm 2) on liquid cycle. When the
medium is removed from the autoclave, swirl it gently to distribute the melted agar or agarose
evenly throughout the solution. Be careful! The fluid may be superheated and may boil over
when swirled.
Allow the medium to cool to 50-60C before adding thermolabile substances (e.g., antibiotics).
To avoid producing air bubbles, mix the medium by swirling. Plates can then be poured directly
from the flask; allow approx. 30-35 ml of medium per 90-mm plate. To remove bubbles from
medium in the plate, flame the surface of the medium with a Bunsen burner before the agar or
agarose hardens. Set up a color code (e.g., two red stripes for LB-ampicillin plates; one black
stripe for LB plates, etc.) and mark the edges of the plates with the appropriate colored
markers.
When the medium has hardened completely, invert the plates and store them at 4C until
needed. The plates should be removed from storage 1-2 hours before they are used. If the

plates are fresh, they will "sweat" when incubated at 37C. When this condensation drops on
the agar/agarose surface, it allows bacterial colonies or bacteriophage plaques to spread
and increases the chances of cross-contamination. This problem can be avoided by wiping off
the condensation from the lids of the plates and then incubating the plates for several hours at
37C in an inverted position before they are used. Alternatively, remove the liquid by shaking
the lid with a single, quick motion. To minimize the possibility of contamination, hold the open
plate in an inverted position while removing the liquid from the lid.
NaCl
To prepare a 5 M solution: Dissolve 292 g of NaCl in 800 ml of H2O. Adjust the volume to 1
liter with H2O. Dispense into aliquots and sterilize by autoclaving. Store the NaCl solution at
room temperature.
NaOH
The preparation of 10 N NaOH involves a highly exothermic reaction, which can cause
breakage of glass containers. Prepare this solution with extreme care in plastic beakers. To
800 ml of H2O, slowly add 400g of NaOH pellets, stirring continuously. As an added
precaution, place the beaker on ice. When the pellets have dissolved completely, adjust the
volume to 1 liter with H2O. Store the solution in a plastic container at room temperature.
Sterilization is not necessary.
Potassium Acetate
5 M potassium acetate, 60 ml
glacial acetic acid, 11.5 ml
H2O, 28.5 ml
The resulting solution is 3 M with respect to potassium and 5 M with respect to acetate. Store
the buffer at room temperature.
Rich medium
LB
YT
Terrific Broth
For solid medium, please see Media Containing Agar or Agarose.
SDS
Also called sodium lauryl sulfate. To prepare a 20% (w/v) solution, dissolve 200 g of
electrophoresis-grade SDS in 900 ml of H2O. Heat to 68C and stir with a magnetic stirrer to
assist dissolution. If necessary, adjust the pH to 7.2 by adding a few drops of concentrated
HCl. Adjust the volume to 1 liter with H2O. Store at room temperature. Sterilization is not
necessary. Do not autoclave.
STE
10 mM Tris-Cl (pH 8.0)
0.1 M NaCl
1 mM EDTA (pH 8.0)
Sterilize by autoclaving for 15 minutes at 15 psi (1.05 kg/cm 2) on liquid cycle. Store the sterile
solution at 4C.

TE
100 mM Tris-Cl (desired pH)
10 mM EDTA (pH 8.0)
(10x Tris EDTA) Sterilize solutions by autoclaving for 20 minutes at 15 psi (1.05 kg/cm 2) on
liquid cycle. Store the buffer at room temperature.
Terrific Broth
deionized H2O, to 900 ml
tryptone, 12 g
yeast extract , 24 g
glycerol, 4 ml
For solid medium, please see Media Containing Agar or Agarose.
Shake until the solutes have dissolved and then sterilize by autoclaving for 20 minutes at 15
psi (1.05 kg/cm2) on liquid cycle. Allow the solution to cool to 60C or less, and then add 100
ml of a sterile solution of 0.17 M KH2PO4, 0.72 M K2HPO4. (This solution is made by dissolving
2.31 g of KH2PO4 and 12.54 g of K2HPO4 in 90 ml of deionized H2O. After the salts have
dissolved, adjust the volume of the solution to 100 ml with deionized H2O and sterilize by
autoclaving for 20 minutes at 15 psi [1.05 kg/cm2] on liquid cycle.)
Tris-Cl
Dissolve 121.1 g of Tris base in 800 ml of H2O. Adjust the pH to the
desired value by adding concentrated HCl.
pH HCl
7.4 70 ml
7.6 60 ml
8.0 42 ml
(1 M) Allow the solution to cool to room temperature before making final adjustments to the pH.
Adjust the volume of the solution to 1 liter with H2O.
Dispense into aliquots and sterilize by autoclaving.
If the 1 M solution has a yellow color, discard it and obtain Tris of better quality. The pH of Tris
solutions is temperature-dependent and decreases approx. 0.03 pH units for each 1C
increase in temperature. For example, a 0.05 M solution has pH values of 9.5, 8.9, and 8.6 at
5C, 25C, and 37C, respectively.
YT
deionized H2O, to 900 ml
tryptone, 16 g
yeast extract, 10 g
NaCl, 5 g
For solid medium, please see Media Containing Agar or Agarose.
To prepare 2x YT medium, shake until the solutes have dissolved. Adjust the pH to 7.0 with 5
N NaOH. Adjust the volume of the solution to 1 liter with deionized H2O. Sterilize by
autoclaving for 20 minutes at 15 psi (1.05 kg/cm 2) on liquid cycle.