Isolation of Genomic DNA from Escherichia coli cells

Genomic DNA is a large molecule and is prone to damage while isolation. DNases produced by cells can damage the DNA, which is prevented by the addition of Ethylene diamine tetraacetate (EDTA) in the buffer used. When the mechanical stress and shear forces are eliminated, fragments of 20-­50 Kb are obtained routinely.

Extraction and purification of genomic DNA have four basic steps:

1. Growth of bacterial culture.

2. Harvesting and lysing the cells.

3. Separation and purification of genomic DNA from other components.

4. The concentration of the genomic DNA

Isolated genomic DNA can be visualized by performing electrophoresis with agarose gel with 0.8­0.9% of agarose.

PRINCIPLE

Isolation of genomic DNA is possible only after lysing the cells. Cells are harvested in a solution containing Tris buffer, Sodium salts, EDTA, and Sodium dodecyl sulfate (SDS). Hypotonic conditions are used to lyse the cells. EDTA is used in the buffer to chelate Magnesium ions (Mg2+) that are required for the action of nucleases that are produced by the bacterial cells thus protecting the DNA. EDTA also increases the permeability of the membrane to SDS. Sodium ions give ionic strength to the buffer and stabilize DNA during isolation. SDS is an anionic detergent that induces lysis of cells by removing the phospholipid bilayer and denaturing proteins. Thus the cell wall is broken down and the protein lysate is solubilized by heating with SDS. The process is carried out at pH ­ 8.0 and heating at 68ºC ensures complete lysis. A mixture of DNA and RNA is separated from proteins and other cell components by extraction with organic solvents – phenol and chloroform in the ratio of 1:1. DNA is precipitated using ethanol, which dehydrates the DNA. The DNA is then re­suspended in a buffer with EDTA or in sterile double distilled water.

REAGENTS REQUIRED:

1.  1 M Tris: Tris base (12.11 g) was added to 80 ml of water. pH was adjusted to 8.0 by adding concentrated HCl. The volume was made up to 100 ml. This was autoclaved and stored at room temperature.
2. 0.5 M EDTA.2H2O: Disodium EDTA. 2H2 O (18.61 g) was added to 80 ml of water and stirred well with a magnetic stirrer. The pH was adjusted to 8.0 with Sodium hydroxide (1N) solution. The volume was made up to 100 ml with distilled water. This was autoclaved and stored at room temperature.
3. 2% SDS: SDS (0.8 g) was dissolved in 8 ml of water and stirred with a magnetic stirrer and made up to 40 ml with distilled water.
4. STE­ 1
   
   

   Reagent	Stock concentration	Required concentration	Required volume
   Tris buffer (pH 8.0)	1 M	100 mM	1 ml
   EDTA	0.5 M	1 mM	200 µl
   NaCl solution	-	100 mM	600 µg in 98.8 ml of water

   
   
5. STE­ 2
   
   

   Reagent	Stock concentration	Required concentration	Required volume
   Tris buffer (pH 8.0)	1 M	10  mM	1 ml
   EDTA	0.5 M	50  mM	1 ml
   NaCl solution	-	100 mM	600 µg in 98.8 ml of water

   
   
6. Tris EDTA ( 10X Tris EDTA, (pH 8.0)

   Reagent	Stock concentration	Required concentration	Required volume
   Tris chloride	1 M	100 mM	10  ml
   EDTA	0.5 M	10  mM	2 ml
   Water	-	-	88 ml

   
   

MATERIALS REQUIRED:

E.coli­ was overnight grown culture, 2% SDS, Absolute alcohol, 70% ethanol, Chloroform, sterile tips, double distilled water, Luria Bertani (LB) broth.

PROCEDURE:

• E.coli colonies were picked from preserved slants and were streaked on Luria Bertani (LB) agar to obtain isolated colonies. A single colony was picked and inoculated in 5ml of LB broth. This was incubated at 37ºC, 220 rpm overnight.
• The optical density of this culture was measured at 600 nm and recorded.
• 1.5 ml of this culture was taken in an Eppendorf tube and centrifuged at 10,000 rpm for 10 minutes.
• The supernatant was discarded and the pellet was re­suspended in 500 µl of STE­1 and centrifuged at 8000 rpm for 10 minutes.
• The supernatant was discarded. To the pellet 500 µl of STE­2 was added followed by 125 µl of 2% SDS. This was mixed thoroughly and incubated at 68ºC for 20 minutes.
• To the tubes, 500 µl of chloroform was added and centrifuged at 9000 rpm for 10 minutes.
• The supernatant was then collected in another Eppendorf tube without disturbing the pellet.
• To this Eppendorf, an equal volume of 100% of Isopropyl alcohol (IPA) was added and left at room temperature for 10-­20 minutes.
• The tubes were then centrifuged at 9000 rpm for 10 minutes. The supernatant was discarded and to the pellet, 200 µl of 70% ethanol was added.
• The tubes were centrifuged at 9000 rpm for 10 minutes.
• The pellet obtained was then air-dried, suspended in 20­-25 µl of double-distilled water, and stored at ­20ºC until further use.