EXPERIMENT NO. 1
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...
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