Plasmid Isolation - Alkaline Lysis Method

Aim: To extract plasmid from bacteria using alkaline lysis method.

Theory: The most widely used method of extracting plasmid from cells is by using
chemical treatment. The method takes advantage of the fact that genomic DNA gets
denatured at pH 11.5 -12, but plasmid DNA does not. The cells are gently lysed
using alkaline solution, then neutralizing the released contents using a high
concentration of potassium acetate (Refer to section 4.1 of Working with DNA).
Following extraction, phenol chloroform extraction could be performed to purify the
DNA. The plasmid in the supernatant is precipitated using ethanol (100%) (Refer to
section 3.5 of Working with DNA). The extracted plasmid could be stored in water
or TE buffer which maintains the integrity of the plasmid.

Materials Required:

Glassware: Test tube, measuring cylinder, thermometer.

Plastic ware: 1.5 ml Microfuge tube, Test tube stand, Float, Micro pipettes (1 ml,
200µl, 20µl).

Reagents: LB broth, Antibiotic (in required concentration), Glucose, 0.5 M EDTA

Misc: Toothpicks, shaking incubator (at 37 ˚C), table top centrifuge machine, water
bath (95˚C), crushed ice.

Prepare 0.5 M EDTA (pH 8.0); adjust the pH using NaOH pellets and 10N NaOH;
Sterilize by autoclaving.

Prepare 1 M Tris-Cl (pH 8.0); adjust the pH using Conc. HCl; Sterilize by
autoclaving.

Solution I (GET buffer): Glucose (50 mM), EDTA (10mM), Tris-Cl (25mM);
Autoclavable

Solution II (freshly prepared): NaOH (0.2 N), SDS (1%)

Solution III (10 ml): Potassium acetate (5ml), Glacial acetic acid (1.2ml), Water
(3.8ml) [pH should be 4-5 – 5.0]

Solution IV (10ml): Tris buffered phenol (5ml), Chloroform:Isoamyl alcohol (5ml)

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Prepare 3M sodium acetate (pH 5.2); adjust the pH by glacial acetic acid. Sterilize
by autoclaving.

Protocol

1. Inoculate 5-10 ml of LB containing the appropriate antibiotic with a single

2. Take 1.5ml culture in microfuge tube and centrifuge at 10000 rpm for 30s to
pellet the cells. Discard the supernatant without disturbing the pellet. Repeat
the step with an additional 1.5ml of culture centrifuged into the same tube.
3. Resuspend the pellet (by vortexing) in 100 µl of ice cold solution I. Incubate at
room temperature for 5 min.
4. Add 200µl of freshly prepared solution II and mix by inverting the tubes 2- 3
times. Incubate on ice for 5 minutes.
( Close the tube and mix the contents by inverting. Make sure that the entire
surface of the tube comes in contact with solution II. Do not vortex or
increase the time duration, it will damage the DNA. )
5. Add 300 µl of ice cold potassium acetate Solution III and mix gently.
Incubate on ice for 5 minutes.
( There should no longer be two distinguishable liquid phases. A flocculent
white precipitate should form. )
6. Centrifuge at 10,000 rpm for 10 min (4 ̊C) and transfer the supernatant to a
new tube.
7. Add 7 µl of 0.5 mg/ml RNase A (Diluted from the stock of 50 mg/ml ).
Incubate at 37 ̊C for 30 minutes in water bath.
8. Add 700 µl of solution IV. Mix the tube and centrifuge at 10,000 rpm/ 10
minutes (4 ̊C). Transfer the aqueous supernatant to a new tube.
(Phenol is corrosive in nature, handle with care)
9. Add equal volume of chloroform:isoamyl alcohol (24:1). Mix well and
centrifuge at 10,000 rpm/ 5 minutes (4 ̊C). Transfer the aqueous supernatant
to a new tube.
10. Add two volumes of ice –cold 100% ethanol. (In addition, add 1/10th volume of
3M sodium acetate solution for better precipitation of DNA). Incubate at -20
℃ minimum for 30-45 minutes or overnight.
11. Centrifuge at 10,000 rpm/ 15 minutes (4 ℃). Decant the supernatant and
invert the tube to allow last drops of supernatant to drain away.

12. Wash the pellet with 500 µl of 70% alcohol. Centrifuge at 10,000 rpm/ 15
minutes (4 ℃). Decant the supernatant and air-dry the pellet briefly (~5-7
min). Do not over-dry the pellet, else it may take a long time to dissolve in the
next step.

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 13. Resuspend the pellet containing plasmid DNA in 50 µl water (or TE). (In case
the pellet does not dissolve leave it in incubator at 37 C
̊ for 15 mins. Store at
minus -20 C̊ ). The extracted plasmid can be visualized on a gel.

Extra facts:

Solution I is used to destabilize the cell wall and cell membrane.

Solution II increases the pH causing cell lysis and the genomic DNA to denature.
SDS acts as detergent.

Solution III neutralizes the pH causing the genomic DNA to clump along with cell
debris.

Phenol chloroform extraction : Proteins in an aqueous phase are folded so that their
nonpolar surfaces are hidden, but in phenol they can unfold and still be soluble.
Forming an emulsion of the phenol and water by vigorous mixing will tend to
partition the nucleic acids into the aqueous phase and protein into the organic
phase. Thus, leading to their separation. Excess phenol is removed using chloroform
:isoamyl alcohol wash after the purification step.

Isopropanol/Ethanol: Lowers the dielectric point causing the DNA to precipitate.