Plasmid Extraction
Plasmid Extraction
Plasmid Extraction
Protocol
In this protocol, plasmid DNA is isolated from small-scale (12 mL) bacterial cultures. Yields vary
between 100 and 5 g of DNA, depending on the copy number of the plasmid. Miniprep DNA is
sufciently pure for use as a substrate or template in many in vitro enzymatic reactions. However,
further purication is required if the plasmid DNA is used as the substrate in sequencing reactions.
MATERIALS
It is essential that you consult the appropriate Material Safety Data Sheets and your institutions Environmental
Health and Safety Ofce for proper handling of equipment and hazardous material used in this protocol.
RECIPES: Please see the end of this protocol for recipes indicated by <R>. Additional recipes can be found online at
http://cshprotocols.cshlp.org/site/recipes.
Reagents
Alkaline lysis solution I <R>, ice cold
Alkaline lysis solution II <R>
Alkaline lysis solution II should be freshly prepared and used at room temperature.
Equipment
Incubator shaker, preset to 37C
KimWipes
From the Molecular Cloning collection, edited by Michael R. Green and Joseph Sambrook.
2016 Cold Spring Harbor Laboratory Press
Cite this protocol as Cold Spring Harb Protoc; doi:10.1101/pdb.prot093344
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METHOD
Preparation of Cells
1. Inoculate 2 mL of rich medium (LB, YT, or Terric Broth) containing the appropriate antibiotic
with a single colony of transformed bacteria. Incubate the culture overnight at 37C with vigorous
shaking.
To Ensure That the Culture Is Adequately Aerated:
i. The volume of the culture tube should be at least four times greater than the volume of the
bacterial culture.
ii. The tube should be loosely capped.
iii. The culture should be incubated with vigorous agitation.
2. Pour 1.5 mL of the culture into a microcentrifuge tube. Centrifuge at maximum speed for 30 sec
at 4C in a microcentrifuge. Store the unused portion of the original culture at 4C.
3. When centrifugation is complete, remove the medium by aspiration, leaving the bacterial pellet
as dry as possible.
This step can be conveniently accomplished with a disposable pipette tip or Pasteur pipette attached to a
vacuum line and a side arm flask (see Fig. 1). Use a gentle vacuum, and touch the tip to the surface of the
liquid. Keep the tip as far away from the bacterial pellet as possible as the fluid is withdrawn from the tube.
This minimizes the risk that the pellet will be sucked into the side arm flask. Alternatively, remove the
supernatant using a pipettor or Pasteur pipette and bulb. Use the pipette tip to vacuum the walls of the
tube to remove any adherent droplets of fluid. The penalty for failing to remove all traces of medium from the
bacterial pellet is a preparation of plasmid DNA that is resistant to cleavage by restriction enzymes. This is
because cell-wall components in the medium inhibit the action of many restriction enzymes. This problem
can be avoided by resuspending the bacterial pellet in ice-cold STE (0.25 volume of the original bacterial
culture) and centrifuging again.
Lysis of Cells
4. Resuspend the bacterial pellet in 100 L of ice-cold Alkaline lysis solution I by vigorous vortexing.
Make sure that the bacterial pellet is completely dispersed in Alkaline lysis solution I. Vortexing two micro-
centrifuge tubes simultaneously with their bases touching increases the rate and efficiency with which the
bacterial pellets are resuspended. The original protocol (Birnboim and Doly 1979) called for the use of
Gentle suction
Pellet
Vacuum line
Vacuum traps
FIGURE 1. Aspiration of supernatants. Hold the open microcentrifuge tube at an angle, with the pellet on the upper
side. Use a disposable pipette tip attached to a vacuum line to withdraw fluid from the tube. Insert the tip just beneath
the meniscus on the lower side of the tube. Move the tip toward the base of the tube as the fluid is withdrawn. Use
gentle suction to avoid drawing the pellet into the pipette tip. Keep the end of the tip away from the pellet. Finally,
vacuum the walls of the tube to remove any adherent drops of fluid.
Minipreps
lysozyme at this point to assist in dissolution of the bacterial cell walls. This step can be safely omitted when
dealing with bacterial cultures of <10 mL in volume.
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 ve times. Do not vortex! Store the
tube on ice.
Make sure that the entire surface of the tube comes in contact with Alkaline lysis solution II.
Prolonged exposure of superhelical circular DNA to alkali results in irreversible denaturation (Vinograd
and Lebowitz 1966). The resulting cyclic coiled DNA, which cannot be cleaved with restriction
enzymes, migrates through agarose gels at about twice the rate of superhelical, closed-circular DNA
and stains poorly with SYBR dyes. Traces of this condensed form of closed-circular DNA can often
be seen in plasmids prepared by alkaline lysis. The presence of a small amount of cyclic coiled DNA
may be aesthetically unpleasant but is generally harmless. The easiest way to eliminate formation
of cyclic coiled DNA in plasmid preparations is to follow the directions for preparing alkaline lysis
buffers to the letter. Alternatively, arginine buffer may be used in place of standard Alkaline lysis buffer II
in Step 5.
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
for 35 min on ice.
7. Centrifuge the bacterial lysate at maximum speed for 5 min at 4C in a microcentrifuge. 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 min at 4C in a micro-
centrifuge. Transfer the aqueous upper layer to a fresh tube.
Some investigators find the extraction with phenol:chloroform to be unnecessary. However, the elimination
of this step sometimes results in DNA that is resistant to cleavage by restriction enzymes. The purpose of
extracting with chloroform is to remove residual phenol from the aqueous phase. Phenol is slightly soluble in
H2O, but it can be displaced into the organic phase by chloroform.
11. Remove the supernatant by gentle aspiration as described in Step 3 above. Stand the tube in an
inverted position on a paper towel to allow all of the uid to drain away. Use a KimWipe or
disposable pipette tip to remove any drops of uid 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 min at 4C in a microcentrifuge.
13. Again remove all of the supernatant by gentle aspiration as described in Step 3.
Take care with this step because 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 uid is visible in the tube (510 min).
If the pellet of DNA is dried in a desiccator or under vacuum, it becomes difficult to dissolve under some
circumstances and may denature (Svaren et al. 1987). Drying the pellet for 1015 min at room temperature
is usually sufficient for the ethanol to evaporate without the DNA becoming dehydrated.
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.
Any problems encountered during plasmid DNA preparation are evident upon analysis of the DNA follow-
ing restriction enzyme digestion. See Troubleshooting.
TROUBLESHOOTING
Problem (Step 15): Very little or no DNA is visible on the gel either before or after restriction digestion.
Solution: The pellet of nucleic acid was likely inadvertently discarded after ethanol precipitation in
Step 11. Be sure to remove the ethanol by gentle aspiration as soon as possible after the centri-
fugation step. If the centrifuge tube is left to stand for too long, the pellet of DNA will become
detached from the wall.
However, as a quick alternative to starting over, extract the nal DNA preparation
with phenol:chloroform; recover by standard ethanol precipitation and subsequent washing
in 70% ethanol. Alternatively, perform the restriction digest in a larger volume (100200 L),
using vefold more enzyme. At the end of the digestion, recover the DNA by standard ethanol
precipitation.
Sterilize each batch of TE by autoclaving, and dispense 1-mL aliquots into sterile microcen-
trifuge tubes (or sterilize TE in small aliquots). Use a fresh aliquot every day.
Try to maintain sterile technique when using stock solutions.
If bacterial DNase copuries with the plasmid DNA, extract the DNA with phenol:chloroform,
recover by standard ethanol precipitation, and resuspend in fresh TE.
Minipreps
RECIPES
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 min at
15 psi (1.05 kg/cm2) on liquid cycle, and store at 4C.
For plasmid preparation.
STE
10 mM Tris-Cl (pH 8.0)
0.1 M NaCl
1 mM EDTA (pH 8.0)
Sterilize by autoclaving for 15 min at 15 psi (1.05 kg/cm2) on liquid cycle. Store the sterile solution
at 4C.
TE Buffer, 10
100 mM Tris-Cl (desired pH)
10 mM EDTA (pH 8.0)
Sterilize solutions by autoclaving for 20 min at 15 psi (1.05 kg/cm2) on liquid cycle. Store the buffer
at room temperature.
Terric Broth
YT
Tryptone, 16 g
Yeast extract, 10 g
NaCl, 5 g
Deionized H2O, to 900 mL
To prepare 2 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/cm2) on liquid cycle.
REFERENCES
Birnboim HC, Doly J. 1979. A rapid alkaline extraction procedure for Svaren J, Inagami S, Lovegren E, Chalkley R. 1987. DNA denatures upon
screening recombinant plasmid DNA. Nucleic Acids Res 7: 15131523. drying after ethanol precipitation. Nucleic Acids Res 15: 87398754.
Cloninger C, Felton M, Paul B, Hirakawa Y, Metzenberg S. 2008. Control of Vinograd J, Lebowitz J. 1966. Physical and topological properties of circular
pH during plasmid preparation by alkaline lysis of Escherichia coli. Anal DNA. J Gen Physiol 49: 103125.
Biochem 378: 224225.
Paul B, Cloninger C, Felton M, Khachatoorian R, Metzenberg S. 2008. A
nonalkaline method for isolating sequencing-ready plasmids. Anal
Biochem 377: 218222.
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