Isolation of Genomic DNA from E. coli DH5α cells.

Biological

Overnight grown culture of E. coli.

Chemicals

A. Luria Broth medium to grow E. coli: Tryptone - 1.0g Yeast extract - 0.5g NaCl - 0.5g Glucose - 0.1g Dissolve the components in 75ml of distilled water, adjust the pH to 7.6 and finally make the volume to 100ml. Sterilize by autoclaving

B. GTE mix: 50mM Glucose 50mM Tris-HCl 10mM EDTA Sterilize by autoclaving

C. 10mM NaCl Sterilize by autoclaving

D. 10% SDS Dissolve 10g of SDS in 80 ml of distilled water adjust pH to 7.2. Raise the volume to 100ml. Do not Autoclave.

Glassware/Plastic ware

All glassware and plastic ware used should be sterilized:

1. Conical flasks 100 ml
2. plates
3. Eppendroffs
4. Micro tips
5. Pipettes

Instruments/ equipment

1. Orbital shaker
2. pH meter
3. Micropipettes
4. Weighing balance
5. Centrifuge

Isolation of genomic DNA

The isolation of genomic DNA is important as it serves as one of the important starting materials of many further experiments, such as PCR, restriction digestion and whole genome sequencing, etc. There are several basic steps in DNA extraction. The five steps are as follows:

1. Homogenization or disruption of cells

   The cell must first be lysed (broken open) to release the nucleus in eukaryotes or nuleoid in prokaryotes. Cells are broken by grinding, tissue homogenization, or treatment with Iysozyme

2. Inhibition of DNAase:

   At this point the DNA must be protected from enzymes that will degrade it, causing shearing. Many of the nucleases present in cells can digest nucleic acids. When the cell is disrupted, the nucleases can cause extensive hydrolysis. Nucleases apparently present on human fingertips are notorious for causing spurious degradation of nucleic acids during purification. Chelating agents are added to remove metal ions required for nuclease activity.

3. Dissociation of nucleoprotein complexes

   DNA-protein interactions are disrupted with SDS, phenol, or broad spectrum proteolytic enzymes as pronase or proteinase K. Alkaline pH and high concentration of salts improve the efficiency of the process.

4. Removal of contaminating materials

   Contaminating molecules especially proteins are removed by treatment with phenol or chloroform-isoamyl alcohol or phenol chloroform. Proteins can also be removed by salting out proteins by sodium acetate.

5. Precipitation of DNA

   Once the DNA is released, it must be precipitated in alcohol (with salt). The DNA in the aqueous phase is precipitated with cold (0oC) ethanol. The precipitate is usually redissolved in buffer and treated with phenol or organic solvent to remove the last traces of protein, followed by reprecipitation with cold ethanol. RNA is removed by limited treatment with deoxyribonuclease-free ribonuclease.

6. Washing of DNA:

   Final step is the washing of DNA with 70% ethanol to remove the traces of salt used in precipitation step. This is followed by drying and dissolving in double distilled water or TE buffer.

1. Take 1.5 ml of overnight grown culture of E. coli in microfuge tube.
2. Centrifuge at 10,000 rpm for 5 minutes to obtain pellet
3. Incubate at room temperature for 5 minutes.
4. Add 400 μl of 1% SDS and at room temperature for 5 minutes.
5. Add 60 μl of 10mM NaCl and 700 μl of isopropanol in ice.
6. Invert mix.
7. Centrifuge at 10,000 rpm for 20 minutes.
8. Dissolve in 50 μl of TE buffer/ddH2O.
9. Add the loading buffer and subject it to gel electrophoresis.

Animation of the experiment:--

(PASTE THE WELL LABELLED GEL PHOTOGRPAPH)

Nucleic acids are the most polar of the biopolymers and are therefore soluble in polar solvents and precipitated by nonpolar solvents. It is also imperative to mention here, that the source of DNA isolation is an important consideration for the protocol to be followed and the quality as well as the purity of the product obtained. The extracted DNA was dissolved in TE buffer. The quality of DNA was judged by the electrophoresis. On electrophoresis one band was observed near the well which clearly indicate that the band is of high molecular weight and thus it's a genomic DNA. Since there only one band is seen on the gel, so no shearing of DNA has taken place.

1. Use caution when operating the centrifuge.
2. The laboratory surfaces should be very clean during all procedures used in this activity.
3. Use thoroughly clean instruments and glassware. Rinse all equipment with isopropyl alcohol or acetone.
4. Ethanol is highly flammable; use caution.