Isolation of Genomic DNA from Bacterial Cells

To isolate genomic DNA from the bacterial cells and visualizing the same DNA by gel electrophoresis.

Introduction
Genomic DNA preparation differs from the plasmid DNA preparation. Genomic DNA is extracted from bacterial cells by immediate and complete lysis whereas plasmid DNA is isolated by slow-cell lysis to form a sphacroplast.

Principle
The procedure of genomic DNA extraction can be divided into 4 stages:

1. A culture of bacterial cell is grown and harvested.
2. The cells are broken open to release their contents.
3. The cells extracted are treated to remove all components except the DNA.
4. The resulting DNA is then controlled.
   The cell is lysed by adding guanidium thiocyanate and a detergent comprising solution A. It is then centrifuged to separate the RNA and proteins. The resulting supernatant mainly consists of genomic DNA and sometimes RNA. The DNA is precipitated using alcohol.

Materials

• Materials to be stored at –20°C: Bacterial cell pellet (in a 1.5-mL tube).
• Materials to be stored at 40°C: Solution A, Solution B, and control DNA (run only 1 control along with 5 samples).
• Materials to be kept at room temperature: 1.5-mL capillaries, dispenser with tubing ethanol; microcentrifuge, Irans illuminator.

Storage and Handling

1. Store material according to the labeled temperatures in refrigerator, freezer compartments, or room temperature.
2. Store bacterial pellets in freezer/freezer compartments of the fridge on arrival.
3. Handle solution A with care, as it is corrosive in nature.
4. All the reagents are stable for a period of 4 months if stored under recommended conditions.

Procedure

1. Thaw bacterial cells to room temperature.
2. Resuspend the cells in 700 mL of solution A at room temperature.
3. Stand at RT for 5 minutes and spin for 10 minutes at 10000 rpm.
4. Collect 500 mL of supernatant. Avoid decanting the pellet.
5. Add 1 mL of distilled ethanol (1000 mL) to 500 mL of supernatant. The tubes were mixed by inverting until white strands of DNA were visibly precipitating.
6. Spin for 4 minutes at maximum speed and the supernatant or spool precipitate DNA is discarded with the help of a pipette tip and transferred into a fresh tube.
7. Wash the DNA pellet with 95% alcohol and, again, add ethanol and decant. Repeat washes and do a final wash with 75% ethanol. Air dry for 5 minutes.
8. Add 100 mL of solution B and incubate for 5 minutes at 55.66°C to increase the stability of genomic DNA.
9. Spin 10 minutes at maximum speed to remove insoluble proteins. The supernatant is pipetted out into a fresh tube.
10. Take 25 mL of freshly extracted DNA and add 10 mL of gel loading dye and load into the wells.
11. Load 10 mL of control DNA and electrophorize along with experimental samples in 1% agarose gel. (Two controls can be run, along with ten samples).

Preparation of 1% Agarose Gel and Electrophoresis

1. Prepare 1X TAE by diluting the appropriate amount of 50X TAE buffer with distilled water.
2. Add 0.5 gm of agarose to 5 mL of 1X TAE in a 250-mL conical flask. Boil to dissolve agarose and cool to a warm liquid.
3. Place the combs of the electrophoresis set such that the comb is about 2-cm away.
4. When the agarose gel temperature is around 600°C, pour the cooled agarose solution in the gel tank. Make sure that the agarose gel is poured only in the center part of the gel tank and is 0.5–0.9 cm thick, without air bubbles.Keep the set undisturbed until the agarose solidifies.
5. Once the gel is solidified, pour 1X TAE buffer slowly into the gel until the buffer level stands at 0.5 to 0.8 cm above the gel surface.
6. Form wells by gently lifting the comb.
7. Connect power cords in a way so that the red cord is with the red electrode and the black cord is with the black electrode. Power should not be switched on before loading.
8. Load the samples into the wells, recording which samples are loaded into which wells as lane 1, 2, etc. Start the power concentration after loading, with the voltage set to 50 V.
9. Run the gel until the second dye from the well has reached 3 4 th of the gel. Use stained dye for staining the gel after electrophoresis.
10. The staining dye used has been diluted in the ratio 1:6 units of distilled water, before use.

Visualizing DNA

1. After the run is completed, switch off the power supply and disconnect the cords.
2. Slowly remove the gel by running a spatula along the walls of the gel tank. Invert the gel onto your palm, ensuring that the palm totally covers the central square. The palm should be held close to the gel to avoid breaking the gel.
3. Use ethidium bromide, put the gel in a small tray, and pour the staining dye on it. Be sure that the gel is completely immersed and the tray is shaken slowly.
4. Place the staining dye in a container and destain the gel by washing with tap water several times until the DNA is visible as a dark band against a light-blue background.

Result
The genomic DNA has been successfully extracted, and when it was run on a gel, the distinct bands were visible.

Interpretation
The molecular weight of control DNA provided was around 50 kb in size. Genomic DNA has a high molecular weight, so it runs along the control DNA. If the shrinking has occurred during extraction, DNA band run below the control DNA. If RNA is present along with extracted DNA, it will be seen between the blue and purple dye.