The main purpose of these slides is to convey information to the Professors, Lecturers, and Students. These slides contain authentic information about this topic which is mentioned in that.

1. ISOLATION OF GENOMIC
DNA FROM BACTERIAL
CELL
M.Phool Badshah

2. INTRODUCTION:
 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 spheroplast.

3. 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.
o 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.

4. 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, trans illuminator.

5. STORAGE AND HANDLING
 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 arrival.
 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 conditions.

6. PROCEDURE
 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 precipitating.
 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. A
 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.
 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 samples).

8. PREPARATION OF 1% AGAROSE GEL AND
ELECTROPHORESIS:
 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 2-cm away.
 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.

9. A
 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.

10. A
 The staining dye used has been diluted in the ratio 1:6
units of distilled water, before use.
 Visualizing DNA:
o After the run is completed, switch off the power supply
and disconnect the cords.
o 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.
o 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.

11. A
 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.
 Results:
o The genomic DNA has been
successfully extracted, and when it
was run on a gel, the distinct bands
were visible.

12. 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.