AIM: To perform isolation of the genomic DNA from the bacterial cell.
Biological: Bacterial culture (DH5α)
Solution 1 - 10ml
•Glucose (50mM) - 500μl
•Tris-Cl (pH 8.0, 25mM) - 250μl
•EDTA (pH 8.0, 10mM) - 200μ
Phenol: chloroform: isoamylalcohol (25:24:1), Absolute ethanol, 70% ethanol, Sterile distilled water Apparatus: Micropipettes, conical flask, measuring cylinder, beaker, gloves.
The procedure for genomic DNA preparation from a culture of bacterial cells (lacking plasmids) can be divided into four stages:- 1. A culture of bacteria is grown and then harvested.
2. The cells are broken open to release their contents.
3. This cell extract is treated to remove all components except the DNA. 4. The resulting DNA solution is concentrated.
Growth of bacterial cells
First of all the bacteria is grown in a liquid medium. The culture medium provides the essential nutrients at concentrations that will allow the bacteria to grow and divide efficiently E.coli cells are grown in LB which is a complex or undefined medium. LB includes:
Preparation of cell extract
The bacterial cell is enclosed in a cytoplasmic membrane and surrounded by a rigid cell wall. With some species, including E.coli the cell wall may itself be enveloped by a second, outer membrane. All of these barriers have to be disrupted to release the cell components. Techniques for breaking open bacterial cells can be divided into •physical methods, in which the cells are disrupted by mechanical means , •chemical methods, where cell lysis is brought about by exposure to chemical agents that affect the integrity of the cell barriers. Chemical lysis generally involves one agent attacking the cell wall and another disrupting the cell membrane. The chemicals that are used depends on the species of bacterium involved, but with E.coli and related organisms, weakening of the cell wall is usually brought about by lysozyme, ethylenediamine tetraacetate (EDTA) or a combination of both. EDTA (present in solution1)
•Removes magnesium ions that are essential for preserving the overall structure of the cell envelope •Inhibits cellular enzymes that could degrade DNA.
SDS (solution 2)
•Aids the process of lysis by removing lipid molecules and thereby cause disruption of the cell membranes.
•Provides an isotonic environment.
Tris-Cl (present in solution 1)
•Acts as a buffer.
Having lysed the cells, the final step in preparation of a cell extract is removal of insoluble cell debris. Components such as partially digested cell wall fractions can be pelleted by centrifugation leaving the cell extract as a reasonably clear supernatant.
Purification of DNA from a cell extract
In addition to DNA, a bacterial cell extract contains significant quantities of protein and RNA.A variety of methods can be used to purify the DNA from this mixture. One approach is to treat the mixture with reagents which degrade the contaminants, leaving a pure solution of DNA. The standard way to deproteinize a cell extract is to add phenol or a 1:1 mixture of phenol and chloroform. These organic solvents precipitate proteins but leave the nucleic acids in aqueous solution. When the layers are separated by centrifugation, precipitated protein molecules are left as a white coagulated mass at the interface between the aqueous and organic layers. The aqueous solution of nucleic acids can then be removed with a pipette. Protease –protease such as proteinase K is used that breaks polypeptides down into smaller units, which are more easily removed by phenols.
Concentration of DNA samples- Ethanol precipitation
In the presence of salt such as sodium ions, and at a temperature of -20oC or less, absolute ethanol efficiently precipitates polymeric nucleic acids. Is has the added advantage of leaving short chain and monomeric nucleic acid components in solution. Ribonucleotides produced by ribonuclease treatment are therefore lost in this stage.
1.1.5ml of E. coli (DH5α) culture was taken in a microfuge tube. 2.It was centrifuged at 6000 rpm for 5 min to pellet down the cells. Supernatant was decanted in a discard beaker. 3.Step 1 and 2 were repeated so the total volume of culture that was used was 3ml. 4.200 μl of solution I was added to the pellet and was mixed with the help of vortex. 5.It was kept at room temperature for 5 minutes.
6.400μl of solution II was added. Gentle mixing was done by inverting the tube 4-6 times. 7.After mixing it was kept in ice for 10 minutes. While opening the cap a thick thread was observed. Thread indicates that cells have been lysed. 8.Equal volume of phenol: chloroform: isoamylalcohol (25:24:1) (i.e. 600μl) was added to the cell lysate. Gentle mixing was done by inverting the tube 5-6 times. 9.It was centrifuged at 13000 rpm for 10 minutes at 4˚C.
10.Upper aqueous phase (containing DNA), middle interphase (containing precipitated proteins), and lower phase (phenol-chloroform) were obtained. 11.Upper phase was taken out into a fresh microfuge tube.
12.Steps 8-11 were repeated.
13.Equal volume of chloroform was added to extract out the remaining traces of phenol. It was gently mixed by inverting the microfuge tube. 14.It was centrifuged at 13000 rpm for 10 minutes at 4˚C.
15.Upper aqueous phase containing DNA and lower phase containing chloroform were obtained. 16.Aqueous phase containing DNA was taken out into a fresh microfuge tube. 17.Sodium acetate (0.1 volume of extract) was added.
18.0.6 volume of isopropanol or 2.5 volume of absolute ethanol was added to precipitate the DNA. Gentle mixing was done. 19.It was centrifuged at 13000rpm for 10 minutes at 4˚C.
20.Supernatant was decanted and 500µl of 70%ethanol was added for washing. 21.It was centrifuged at 13000rpm for 5 minutes at 4ºC.
22.Supernatant was decanted and DNA pellet was kept for drying at 40˚C. 23.After drying DNA pellet became transparent. Pellet was reconstituted in 50µl of sterile distil water at kept at 37˚C for 1-2 hrs. 24.Genomic DNA thus obtained was analyzed by agarose gel electrophoresis.
1.Solutions should be made carefully.
2.This procedure should be carried out at low temperature so that nucleases are inactive. 3.Mixing should be done gently otherwise it can lead to fragmentation of DNA. 4.Centrifuge should be balanced.
5.Only sterile materials should be used.
6.Gloves should be used while handling ethidium bromide.
7.Bacterial culture should be discarded after bleaching.
8.Protective shield should be worn while working with UV.