Isolation of plasmids from the given bacterial culture by alkaline lysis method

Aim: To isolate different forms of plasmids from the given bacterial suspension.

Principle: Joshua Lederberg (1952) coined the term plasmid. Plasmids are extra
chromosomal genetic material that can replicate autonomously. they are used in
molecular biology and biotechnology as cloning and expression vectors. A common
technique in molecular biology is generally referred to as “minipreps”, which usually use
an alkaline lysis method. Minipreps are used to isolate small quantities of plasmid DNA
from bacteria. The bacterial cells lysed by treating alkaline lysis buffer and plasmid are
separated. TENS buffer is commonly used. Basic pH of TRIS buffer helps to denature
DNA, and EDTA (ethylenediaminetetraacetic acid) binds divalent cations such as
calcium and magnesium. Since these ions help maintain the integrity of the cell
membrane, eliminating them with EDTA destabilizes the membrane and inhibit DNases
(enzymes that degrade DNA).
In addition, RNases are also added to degrade the released RNA. Next, the bacteria are
lysed with strong alkali (Sodium Hydroxide (NaOH) and detergent (Sodium Dodecyl
Sulfate (SDS). The SDS detergent solubilizes the phospholipids and proteins of the cell
membrane resulting in cell lysis and the release of the cells contents. The high
concentration of sodium hydroxide denatures genomic and plasmid DNA, as well as
cellular proteins. The cellular DNA becomes linearized, and the strands are separated,
whereas the plasmid DNA is circular and remains topologically constrained (the two
strands, although denatured remain together). Finally, a neutralization buffer of sodium
acetate is added to neutralize the strong alkaline conditions. The addition of
sodium/potassium acetate results in a high salt concentration that leads to the formation
of a white precipitate that consists of SDS, lipids and proteins. In addition, the
neutralization of the solution allows the renaturation of DNA. The large chromosomal
DNA is captured in the precipitate, whereas the small plasmid DNA remains in solution.
The precipitate and chromosomal DNA is removed by centrifugation. Following
centrifugation, the soluble plasmid DNA can be purified from the solution by various
techniques. The most common is to precipitate the DNA with alcohol (ethanol or
 isopropanol) or high salt (ammonium acetate, lithium chloride, sodium chloride or
sodium acetate) and washed with 70% ethanol. Finally, the plasmid is resuspended in MilliQ
water or TE buffer and stored at 4°C. These plasmids are allowed to migrate on agarose
gel electrophoresis. Depending on the structural configuration difference, the three
forms of plasmids are separated i.e. circular, linear, supercoiled.

Materials Required:
1. Bacterial Culture in LB media
2. TENS buffer
TENS buffer
10N NaOH -1ml
20% SDS -1ml (Sodium Dodecyl Sulphate)
1M TRIS -1ml (Tris, or tris(hydroxymethyl)aminomethane)
0.5M EDTA -200μl
Make up to 100ml with double distilled water
3. 3M Potassium Acetate (pH 5.2)
4. Isopropanol
5. Double distilled water
6. Micropipettes
7. Microcentrifuge tubes
8. Cold centrifuge
LB Media
Bacto yeast extract - 5 gm/L
Tryptone - 10gm/L
Sodium Chloride - 10 gm/L
Dissolve and make up to one litre with double distilled water. Adjust the pH to 7.0
and autoclaved.
Protocol:
1) 1.5 ml of overnight grown E.coli culture, containing plasmid (pBluescript) was
taken in a sterile centrifuge tube.
2) The cells were centrifuged at 4000-6000 rpm for 5 minutes at 4°C and the
supernatant was discarded.
3) The cells were resuspended in 400 µl of TENS buffer, mixed by vertexing and
the tube was placed in ice for 10 minutes.
A basic pH Tris buffer, which helps to denature DNA
EDTA (ethylenediaminetetraaceticacid) that binds divalent cations destabilizing the membrane
and inhibiting DNases.
The bacteria are lysed with strong alkali NaOH,
Detergent (Sodium Dodecyl Sulfate) solubilizes the phospholipids and proteins of the cell
membrane resulting in cell lysis and the release of the cells contents.
4) 150 μl of 3M Sodium/potassium acetate (pH 5.2) was added and the tube was
vortexed slightly and placed in ice for 10 minutes. ( Neutralization buffer of
sodium/potassium acetate is added to neutralize the strong alkaline conditions. Salt
(pottasium acetate) reacts with DNA helps in precipitation. It breaks up into K+ and (CH3COO)–
The positively charged K+ ion neutralize negatively charged PO3– of the DNA and neutralize the
charges on the sugar phosphate backbone of the DNA. Hydrophilic nature of DNA helps it to
dissolve in water but by reacting with potassium acetate, DNA becomes less hydrophilic).
The addition of potassium acetate results in a high salt concentration that leads to the
formation of a white precipitate that consists of SDS, lipids and proteins. In addition, the
neutralization of the solution allows the renaturation of DNA. The large chromosomal DNA is
captured in the precipitate, whereas the small plasmid DNA remains in solution. The
precipitate and chromosomal DNA is removed by centrifugation.
5) The tube was centrifuged at 12000 rpm for 15 minutes at 4°C and the
supernatant was transferred to another centrifuge tube carefully.
6) 0.7 volume of isopropanol was added and centrifuged at 12000 rpm for 15
minutes at 4°C and the supernatant was discarded. Plasmid/DNA does not dissolve
easily in ethanol or isopropanol, as they both require a polar solvent such as water to
dissociate. Adding ethanol or isopropanol to a salty solution, therefore, causes the DNA to
pellet and precipitate out of solution
7) The pellet was washed with 500µl of 70% ethanol, centrifuged at 12000 rpm for
10-15 minutes and the supernatant was discarded.
70% ethanol allows the salts to dissolve while minimizing DNA solubility.
 8) The pellet was semi-dried and resuspended in 25 µl of 1×TE buffer or milliQ
water and stored at -20°C until further analysis.

Observation:

Conformation of plasmids isolated

Conclusion: Upon electrophoresis, plasmids can be seen as bright bands when visualized under
UVtransilluminator. When run along with a DNA marker, it will be possible to identify the size
of plasmid DNA isolated from bacteria.

Discussion: Plasmid DNA can assume three conformations circular, linear and supercoiled.
when run on agarose gel, amongst these, the supercoiled DNA assumes the smallest
conformation and thus, will travel the fastest and farthest away from the well. This will be
followed by the linear plasmid DNA, which corresponds to the correct size of the DNA marker.
The circular DNA, because of its conformation, will be the slowest and thus will appear above
the other two bands. It is possible during isolation to have more DNA of one plasmid
conformation.