Protocol – Plasmid isolation by alkaline lysis method (miniprep)

Overview

  • Alkaline lysis method of plasmid isolation was originally developed by Brinboim and Doly (1979).
  • In this procedure, bacteria containing the desired plasmid are harvested from liquid bacterial culture by centrifugation.
  • Suspension of bacteria is made in isotonic solution which is subsequently subjected to lysis by an alkaline solution containing a detergent, Sodium dodecyl sulfate (SDS), and an alkali, Sodium hydroxide (NaOH).
  • While sodium dodecyl sulfate serves to lyse cells and denature proteins, alkaline condition denatures genomic DNA, plasmid DNA and proteins.
  • Lysed cell mixture is further neutralized by Potassium acetate (pH 5.2). This results in renaturation of plasmid and genomic DNA. Since plasmid DNA is covalently closed, it reanneals while genomic DNA form precipitate.
  • Precipitate is separated by high speed centrifugation. Plasmid from the supernatant is recovered by precipitation using isopropanol or ethanol.
See also:

Requirement

  • Reagents
    • Resuspension Buffer (50 mM glucose, 25 mM TrisCl (pH 8.0), 10 mM EDTA, pH 8.0)
    • Lysis solution (0.2 N NaOH, 1% (wt/vol) SDS)
    • Neutralization solution (3 M potassium acetate, pH 4.8)
    • Phenol : Chloroform : Isoamyl alcohol (25 : 24 : 1) solution (Optional)
    • 70% Ethanol
    • Isopropanol
    • Tris – EDTA (TE) (100 mM Tris, 10 mM EDTA, pH 8.0)
    • DNase free RNase (10 mg/ml)
  • Equipment and disposables
    • Microcentrifuge tube
    • Micropipette and tips
    • Ice
    • Gloves

Objective: Isolation of plasmid DNA from 1-3 ml of bacterial culture (E. coli DH5α) by Alkaline lysis method.

Starting material: 3 ml overnight grown culture of E. coli DH5α containing plasmid of interest. (see protocol)

Prior to start:
  • Make sure that neutralization solution is chilled
  • Set the centrifuge for cooling (4°C)
  • Prepare the lysis solution freshly
  • Add DNase free RNase in resuspension buffer

Protocol

Step 1: Pour 1.5 ml overnight grown culture in a microcentrifuge tube. Centrifuge at room temperature (or 4°C) for 60 seconds at 12,000 rpm (or 5,000 rpm for 5 min). Remove the supernatant from the tube completely, leaving the bacterial pellet as dry as possible.
Notes:
  • The yield of plasmid DNA is dependent mainly on the copy number of plasmid. For high copy number plasmid, 1.5 ml culture is sufficient to get good yield of plasmid DNA. However, more culture is required for good yield of low-copy number plasmid.
Tips:
  • To remove the medium completely, decant the medium from the microcentrifuge tube after centrifugation. Invert microcentrifuge tube upside down on a paper towel to remove residual liquid. Gently tap the tube on the paper towel to remove liquid sticking on the sides of the tube.
  • To take more bacterial culture (more than 1.5 ml) for plasmid isolation, repeat the above process by adding more culture in the same microcentrifuge tube. Microcentrifuge tube with 2 ml capacity can also be used.
  • To increase the yield of low copy number plasmid DNA, one can collect the bacterial cells from 5 ml culture by repeating the above process thrice. Lysis may not be efficient in such cases due to high number of bacterial cells. In such situation, double the amount of all reagents (resuspension Buffer, lysis solution, and neutralization solution).
  • Chloramphenicol treatment can be used to amplify low-copy number plasmid.
  • The procedure may be stopped at this point and continued later by freezing the bacterial cell pellets. The bacterial cell pellets can be stored at -20°C for months or at -70°C for years.
Precautions:
  • While harvesting the bacteria, speed of centrifugation and time should be optimized in such a way that the pellet after centrifugation should be loose and at the same time supernatant should be clear. If pellet is tight, it would be difficult to make the suspension of the pellet. Generally above mentioned condition works well.
  • Try to remove medium from the pellet completely. Traces of medium may inhibit some of the sensitive restriction enzymes action. Additionally pellet can be washed with 200 μl of resuspension buffer. Generally washing is not required for miniprep.

Step 2: Add 100 μl ice cold resuspension buffer. Resuspend the bacterial pellet properly by vortexing or by slow rounds of pipetting with a 100 μl micropipette. Incubate on ice for 5 min.

Tips:
  • The procedure can be stopped at this point and continued later by freezing the suspension of bacterial cells. It can be stored at -20 °C for months or at -70 °C for years. We recommend to stop the procedure (if necessary) at this stage instead of previous step as making resuspension of frozen bacterial pellet is not so efficient. If you resuspend the bacterial pellet before freezing, you need to just thaw the content and proceed to next step.
  • To get rid of bacterial RNA, one can supplement resuspension buffer with RNase A (final conc. 100 μg/ml). RNases are very stable and retain their activity for long time under harsh conditions, like high alkaline denaturing condition. RNA are degraded upon lysis of cells by RNase A.

Precautions: Ensure that the bacterial pellet is completely dispersed in resuspension buffer prior to lysis. No cell clumps should be visible before the addition of Lysis Solution. Inefficient lysis results in low yield of plasmid.

Step 3: Add 200 μl of freshly prepared Lysis Solution to bacterial suspension. Close the tube tightly, and mix contents thoroughly by inverting and rolling the sealed tube 4 – 6 times until the solution becomes viscous and slightly clear. Incubate on ice for 3 – 5 min.

Precautions:
  • Do not mix by vortexing or vigorous shaking, as this will result in shearing of genomic DNA.
  • Do not allow the lysis reaction to proceed for more than 5 min. Incubation for longer time results in denaturation of supercoiled plasmid DNA. Denatured plasmid will appear as a ghost band in agarose gel analysis of plasmid.
  • Use freshly made lysis solution. Old lysis solution often contains precipitates which can lead to inefficient lysis.

Step 4: Add 150 μl of chilled Neutralization Solution, mix immediately and thoroughly by inverting and rolling the tube 4 – 6 times. Incubate on ice for 3 – 5 min.

Notes:
  • After addition of neutralization solution, a fluffy white material forms and the lysate becomes less viscous. The precipitated material contains genomic DNA, proteins, cell debris and Potassium Dodesyl Sulphate.
Precautions:
  • Use chilled neutralization solution. Precipitation is enhanced by using chilled neutralization solution and incubating on ice.
  • The lysate should be mixed thoroughly to ensure the complete precipitation of SDS in the form of potassium dodecyl sulphate.

Step 5: Centrifuge the tube at maximum speed (14000 rpm) in a microcentrifuge for 10 min at 4°C. Transfer the supernatant containing plasmid promptly in new microcentrifuge tube.

Precautions: While transferring the supernatant, take care that white precipitate should not come along with supernatant. Supernatant should be centrifuged again as mentioned above if supernatant contains any suspended particle.

Step 6 (Optional) : Extract the supernatant with Phenol:Chloroform:isoamylalcohol solution. This step will remove impurities including protein and lipid contamination from the plasmid preparation. Add equal volume of Phenol:Chloroform:Isoamylalcohol (25:24:1) in the supernatant. Mix by vortexing for 10 sec. Centrifuge at maximum speed at 4°C. Transfer the supernatant to fresh microcentrifuge tube.

Precautions:
  • While transferring the supernatant, take care that no traces of phenol come along with supernatant. Traces of phenol is sufficient to inhibits most enzymatic reactions.
  • Phenol and chloroform are toxic. Follow the safety rules while handling phenol.

Step 7: Add equal volume of isopropanol in the supernatant. Mix it by inverting the tube 4 – 6 times. Centrifuge at maximum speed (14,000 rpm) for 30 min at 25°C. Remove the supernatant completely.

Precautions:
  • Incubation for longer time at room temperature or on ice increases plasmid yield, but also causes salt precipitation.
  • While removing the supernatant, care should be taken as isopropanal precipitated plasmid pellet is loosely attached to surface and invisible in most cases. Careless removal of supernatant often results in loss of plasmid pellet.
Step 8: Add 500 μl of 70% ethanol to the pellet. Close the tube and invert several times. Centrifuge at 14000 rpm (maximum speed) for 5 min at 25°C. Remove the supernatant completely.

Tips: To remove the supernatant, one can decant the supernatant after first centrifugation. Remains of liquid will be sticking on the wall of microcentrifuge tube. A second flash spin is sufficient to collect all the liquid at the bottom which can be removed by pipetting. Air dry the pellet for 5 min.

Precautions:
  • Take care with this step, as the pellet sometimes does not adhere tightly to the tube and lost while removing the supernatant.
  • Do not overdry the pellet. Overdried pellet is difficult to dissolve.
  • Remove the traces of ethanol as it may inhibit some enzyme reactions.
Step 9: Dissolve the pellet in 25 μl sterile double distilled water or TE (pH 8.0).

Tips: To dissolve the pellet, one can vortex the solution gently for a brief period ( 2 – 3 times for 5 seconds) and also can incubate at 37° for ∼20 minutes.

Storage

  • Solution can be stored at 4°C for few days. Store at -20°C for years.

Precautions: Don’t thaw the plasmid repeatedly. This can cause reduction of supercoiled form of plasmid.

Applications

  • The isolated plasmid is suitable for most of our cloning experiments. Often the amount of supercoiled plasmid is comparatively less, therefore, is not suitable for transfection experiments.

References:

Birnboim, H.C., and Doly, J. (1979). A rapid alkaline extraction procedure for screening recombinant plasmid DNA. Nucleic Acids Res. 7(6), 1513-1523. PMID-388356,