To isolate genomic DNA from the bacterial cells and visualizing the same DNA
by gel electrophoresis.
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.
The procedure of genomic DNA extraction can be divided into 4 stages:
- A culture of bacterial cell is grown and harvested.
- The cells are broken open to release their contents.
- The cells extracted are treated to remove all components except the DNA.
- 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.
Storage and Handling
- 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.
- Store material according to the labeled temperatures in refrigerator, freezer
compartments, or room temperature.
- Store bacterial pellets in freezer/freezer compartments of the fridge on
- Handle solution A with care, as it is corrosive in nature.
- All the reagents are stable for a period of 4 months if stored under recommended
Preparation of 1% Agarose Gel and Electrophoresis
- Thaw bacterial cells to room temperature.
- Resuspend the cells in 700 mL of solution A at room temperature.
- Stand at RT for 5 minutes and spin for 10 minutes at 10000 rpm.
- Collect 500 mL of supernatant. Avoid decanting the pellet.
- 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
- 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
- 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
- Add 100 mL of solution B and incubate for 5 minutes at 55.66°C to
increase the stability of genomic DNA.
- Spin 10 minutes at maximum speed to remove insoluble proteins. The
supernatant is pipetted out into a fresh tube.
- Take 25 mL of freshly extracted DNA and add 10 mL of gel loading dye
and load into the wells.
- Load 10 mL of control DNA and electrophorize along with experimental
samples in 1% agarose gel. (Two controls can be run, along with ten
- Prepare 1X TAE by diluting the appropriate amount of 50X TAE buffer
with distilled water.
- 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.
- Place the combs of the electrophoresis set such that the comb is about
- 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.
- 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.
- Form wells by gently lifting the comb.
- 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.
- 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.
- 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.
- The staining dye used has been diluted in the ratio 1:6 units of distilled
water, before use.
- After the run is completed, switch off the power supply and disconnect the
- 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
- 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
- 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.
The genomic DNA has been successfully extracted, and when it was run on a
gel, the distinct bands were visible.
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.