Protocol – Plasmid Isolation by Boiling Method (Miniprep)

Overview

• The boiling lysis method of plasmid isolation is quick and is recommended for the isolation of small plasmids (up to 10 kb). Plasmids larger than 10 kb should be isolated by other methods (e.g., alkaline lysis method)
• The quality of the plasmid isolated by this method is not as good as the plasmid isolated by the alkaline lysis method. However, the quality is good enough for restriction digestion analysis. Its rapidity together with the plasmid quality suitable for restriction digestion analysis makes this a method of choice for screening of a large number of clones during cloning experiments.
• This method is not suitable for isolating plasmids from E. coli endA + strains (e.g., HB101, JM100).
• In this method, the bacterial cells are given a brief heat treatment in a boiling water bath in presence of lysozyme and Triton X-100. Plasmid DNA, due to its small size, comes out from the bacterial cell, whereas genomic DNA remains trapped inside the cell.
• Subsequent high-speed centrifugation separates the plasmid DNA from the rest of the cell debris, which forms a pellet. Pellet is removed and plasmid DNA is recovered by ethanol or isopropanol precipitation method.

REQUIREMENTS

Reagents and solutions
♦ STE solution [8% (w/v) sucrose, 50 mM Tris-HCl (pH 8.0), 50 mM EDTA (pH 8.0)]
♦ STET solution [8% (w/v) sucrose, 50 mM Tris-HCl (pH 8.0), 50 mM EDTA (pH 8.0), 5% (w/v) Triton X-100)]
♦ Lysozyme stock solution [10 mg/mL Lysozyme in 10 mM Tris-HCl (pH 8.0)]
♦ 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 A (10 mg/ml)

Equipment and disposables
♦ Boiling water bath
♦ Benchtop Refrigerated centrifuge
♦ Microcentrifuge tubes
♦ Micropipette and tips
♦ Ice
♦ Gloves

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

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

Prior to start:
♦ Make sure that STE and STET solutions are chilled
♦ Set the centrifuge for cooling (4°C)
♦ Set the water bath for boiling (95 – 100°C)

Protocol:

Step 1: Harvest bacterial cells from 1.5 ml culture
◊ Pour 1.5 ml overnight grown culture in a microcentrifuge tube.
◊ Centrifuge the tube at room temperature (or 4°C) for 5 min at 6000 × g or 1 min at 12000 × g. 
◊ Remove the supernatant from the tube completely, leaving the bacterial pellet as dry as possible.

Note:
◊ The yield of plasmid DNA is dependent primarily on the copy number of the plasmid. For high copy number plasmid, 1.5 ml culture is sufficient to get a good yield of plasmid DNA. However, more culture is required for low-copy-number plasmid.

Tips:
◊ 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. Tap the tube gently 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 tubes with a 2 ml capacity can also be used.
◊ Chloramphenicol treatment can be used to amplify low-copy number plasmid.

Precautions:
◊ While harvesting bacteria, the speed of centrifugation and time should be optimized in such a way that the pellet after centrifugation should be loose enough to be resuspended easily, and at the same time supernatant should be clear. If the pellet is tight, it would be difficult to make the suspension of the pellet. Generally, the above-mentioned condition works well.
◊ Try to remove the medium from the pellet completely. Traces of medium may inhibit some of the sensitive restriction enzyme action.

Step 2 (Optional): Wash the bacterial cells with STE solution
♦ Add 500 μl ice-cold STE solution.
♦ Resuspend the bacterial pellet properly by vortexing or by slow rounds of pipetting.
♦ Centrifuge the tube at room temperature (or 4°C) for 5 min at 6000 × g or 1 min at 12000 × g. 
♦ Remove the supernatant from the tube completely.

Note:
The purpose of this step is to remove traces of culture medium from the bacterial cells, which otherwise can cause inhibition of some sensitive restriction enzyme reaction.

Tip:
Tris – EDTA solution (100 mM Tris, 10 mM EDTA, pH 8.0) can also be used in place of STE solution to wash the pellet.

Step 3: Resuspend bacterial pellet in STET solution
Add 350 μl of STET solution and resuspend the bacterial pellet properly by vortexing or by slow rounds of pipetting.

Precaution:
Make sure that the bacterial pellet is completely dispersed in the STET solution. No cell clumps should be visible before boiling in the water bath. Clumps can cause a low yield of the plasmid.

Step 4: Treat bacterial cells with lysozyme.
Add 25 μl of freshly prepared solution of lysozyme and mix immediately by vortexing for 5 seconds.

Precautions:
The lysozyme will not work efficiently if the solution pH is less than 8.0.

Step 5: Close the cap and place the tube in a boiling water bath for approximately 1 min (40 sec – 60 sec).

Note:
The cap of the microcentrifuge may open due to the air pressure generated during boiling.

Step 6: Clear the lysate by high-speed centrifugation
♦ Centrifuge the tube at maximum speed (≥12,000g) in a microcentrifuge for 10 min at 4°C or room temperature.
♦ Transfer the supernatant containing plasmid promptly in a new microcentrifuge tube.
♦ Alternatively, one can remove the viscous pellet with a sterile toothpick.

Note:
One can centrifuge the tube at room temperature. Centrifugation at 4°C generates a tighter pellet than centrifugation at room temperature. The tight pellet can be removed easily.

Precaution:
While transferring the supernatant or removing the pellet with a sterile toothpick, take care that debris should not come along with the supernatant. The supernatant should be centrifuged again if it contains any suspended particle.

Step 7 (Optional): Extract the supernatant with Phenol : Chloroform : isoamyl alcohol solution.
♦ This step will remove impurities including protein and lipid contamination from the plasmid preparation.
♦ 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.

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

Step 8: Recover plasmid from the supernatant by isopropanol precipitation.
♦ Add an equal volume of isopropanol to the supernatant.
♦ Mix it by inverting the tube 4 – 6 times. 
♦ Centrifuge at maximum speed (≥12,000g) for 30 min at 25°C.
♦ Remove the supernatant completely.

Precautions:
◊ Incubation of the above mix at room temperature or on ice increases plasmid yield but also causes salt precipitation. Above mentioned condition generally gives good quality of plasmid DNA without much salt contamination.
◊ While removing the supernatant, care should be taken as the isopropanol-precipitated plasmid pellet is loosely attached to the surface and is invisible in most cases. Careless removal of supernatant can result in loss of the plasmid pellet.

Step 9: Wash the pellet with 70% ethanol.
♦ Add 500 μl of 70% ethanol to the pellet. Close the tube and invert several times.
♦ Centrifuge at 14,000 rpm (maximum speed) for 10 min at 25°C.
♦ Remove the supernatant completely.

Tip:
◊ To remove the supernatant, one can decant the supernatant after first centrifugation. Remains of liquid will be sticking on 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:
◊ Take care with this step, as the pellet sometimes does not adhere tightly to the tube and gets lost while removing the supernatant.
◊ Do not overdry the pellet. Overdried pellets are difficult to dissolve.
◊ Remove the traces of ethanol as it may inhibit some enzyme reactions.

Step 10: 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 and also can incubate at 37° for ∼20 minutes.

Storage
The solution can be stored at 4°C for a few days. Store at -20°C for years.

Precaution:
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.