Protocol – Plasmid Isolation by Alkaline Lysis Method (Miniprep)

OVERVIEW

• The 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 conditions denature genomic DNA, plasmid DNA, and proteins.
• In the subsequent step, lysed cell mixture is neutralized by potassium acetate (pH 5.2). This results in the renaturation of plasmid and genomic DNA. Since plasmid DNA is covalently closed, it reanneals in correct conformation while genomic DNA forms precipitate due to random annealing of long strands.
  
• The precipitate is separated by high-speed centrifugation.
• Plasmid from the supernatant is recovered by precipitation using isopropanol or ethanol.

REQUIREMENTS

Reagents and solutions
♦ 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)

* The resuspension buffer can be prepared without glucose for cell wall-containing bacteria. In addition, the resuspension buffer can be supplemented with RNase A. Click here to know more about the composition of the resuspension buffer.
** Use Tris-saturated Phenol for plasmid DNA isolation. Do not use water-saturated phenol.

Equipment and disposables
♦ Microcentrifuge tube
♦ Micropipette and tips
♦ Refrigerated centrifuge
♦ 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 (in LB medium) of E. coli DH5α containing plasmid of interest.

Do this in advance:
◊ Make sure that the neutralization solution is chilled
◊ Set the centrifuge for cooling (4°C)
◊ Prepare the lysis solution freshly
◊ Add DNase-free RNase in the resuspension buffer

PROCEDURE

Step 1: Harvesting bacterial cells from overnight-grown liquid culture
◊ Pour 1.5 ml of overnight grown culture into 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.

Note:
The yield of plasmid DNA depends mainly on the copy number of the plasmid. The plasmid yield would be high for high-copy number plasmid but low for low-copy number plasmid. You can take up to 3 ml culture of low-copy number plasmid.

Tips:
1. To remove the medium completely, decant the medium from the microcentrifuge tube after centrifugation. Invert the 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.
2. 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. A Microcentrifuge tube with a 2 ml capacity can also be used.
3. To increase the yield of low copy number plasmid DNA, one can collect the bacterial cells from a 5 ml culture by repeating the above process thrice. Lysis may not be efficient in such cases due to the high number of bacterial cells. In such a situation, double the amount of all reagents (resuspension Buffer, lysis solution, and neutralization solution).
4. Chloramphenicol treatment can be used to amplify low copy number plasmid.
5. 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:
1. While harvesting the bacterial cells, the speed of centrifugation and time should be optimized in a way that allows harvesting of all bacterial cells but at the same time results in a loose pellet that can be resuspended easily in the subsequent step. A clear yellowish (LB medium color) supernatant indicates the complete harvesting of bacterial cells from the culture. If the pellet is tight, it would be difficult to make the suspension of the pellet. Generally, the above-mentioned condition works well.
2. Try to remove the medium from the pellet as much as possible. Traces of medium may inhibit some of the sensitive restriction enzyme’s action. Additionally, the pellet can be washed with 200 μl of resuspension buffer. Generally washing is not required for miniprep.

Step 2: Resuspension of harvested bacterial cells in resuspension buffer (solution I)
◊ Add 100 μl ice-cold resuspension buffer and 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:
1. 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 stopping the procedure (if necessary) at this stage instead of the previous step as making resuspension of frozen bacterial pellets is not so efficient. If you resuspend the bacterial pellet before freezing, you need to just thaw the content and proceed to the next step.
2. To get rid of bacterial RNA, one can supplement a resuspension buffer with RNase A (final conc. 100 μg/ml). RNases are very stable and retain their activity for a long time under harsh conditions, like high alkaline denaturing conditions. RNA is degraded upon lysis of cells by RNase A.

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

Step 3: Lysis of bacterial cells
◊ 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:
1. Do not mix by vortexing or vigorous shaking as this will result in the shearing of genomic DNA.
2. Do not allow the lysis reaction to proceed for more than 5 min. Incubation for a longer time may result in the denaturation of supercoiled plasmid DNA. The denatured plasmid will appear as a ghost band in agarose gel analysis of the plasmid.
3. Use freshly made lysis solution. Old lysis solutions often contain precipitates which can lead to inefficient lysis.

Step 4: Neutralization of lysate to precipitate genomic DNA and other components of cells leaving the plasmid in solution
◊ Add 150 μl of chilled Neutralization Solution, and mix immediately and thoroughly by inverting and rolling the tube 4 – 6 times.
◊ Incubate on ice for 3 – 5 min.

Note:
After the addition of the neutralization solution, a fluffy white material forms and the lysate becomes less viscous. The precipitated material contains genomic DNA, proteins, cell debris, and Potassium Dodecyl Sulphate.

Precautions:
1. Use a chilled neutralization solution. Precipitation is enhanced by using a chilled neutralization solution and incubating on ice.
2. The lysate should be mixed thoroughly to ensure the complete precipitation of SDS in the form of potassium dodecyl sulfate.

Step 5: Separation of supernatant from the precipitate
◊ Centrifuge the tube at maximum speed (14000 rpm) in a microcentrifuge for 10 min at 4°C.
◊ Transfer the supernatant promptly to a new microcentrifuge tube.

Precaution: 
While transferring the supernatant, take care that the white precipitate should not come with the supernatant. The supernatant should be centrifuged again as mentioned above if it contains any suspended particles.

Step 6 (Optional): Extract the supernatant with Phenol:Chloroform:isoamyl alcohol solution.
◊  Add an equal volume of Phenol:Chloroform: Isoamyl Alcohol (25:24:1) in the supernatant.
◊ Mix by vortexing for 10 sec.
◊ Centrifuge at maximum speed at 4°C.
◊ Transfer the supernatant to a fresh microcentrifuge tube.

Note:
This step will remove impurities including protein and lipid contamination from the plasmid preparation.

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

Step 7: Recovery of plasmid DNA from the supernatant by precipitation
◊ Add an equal volume of isopropanol to 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:
1. Incubation for a longer time at room temperature or on ice increases plasmid yield but also causes salt precipitation.
2. While removing the supernatant, care should be taken as isopropanol precipitated plasmid pellet is loosely attached to the surface and invisible in most cases. Careless removal of supernatant often results in loss of plasmid pellet.

Step 8: Washing with 70% ethanol to remove precipitated salt from plasmid DNA
◊ Add 500 μl of 70% ethanol to the pellet.
◊ Close the tube and invert it several times.
◊ Centrifuge at 14000 rpm (maximum speed) for 5 min at 25°C.
◊ Remove the supernatant completely.

Tip: 
To remove the supernatant, one can decant the supernatant after the centrifugation. The remains of the liquid will be sticking to the wall of the 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:
1. Take care with this step, as the pellet sometimes does not adhere tightly to the tube and is lost while removing the supernatant.
2. Do not overdry the pellet. The overdried pellet is difficult to dissolve.
3. 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).

Tip: 
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
The pålasmid solution can be stored at 4°C for a few days. Store at -20°C/-70°C for years.

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