INTRODUCTION
DNA isolation is a process of purification of DNA from sample using a
combination of physical and chemical methods. DNA is extracted from human cells
for a variety of reasons. With a pure sample of DNA we can test a newborn for a
genetic disease, analyze forensic evidence, or study a gene involved in cancer.
Gram-negative bacteria are a group of bacteria that do not retain the crystal violet stain (when rinsed with alcohol) used in the Gram staining method of bacterial
differentiation,[1] making positive
identification possible. It has thin layers of peptidoglycan and the bright
pink to red colour will appear when it stained with safranin. The example of
gram-negative bacteria are Pseudomonas sp. and E.coli sp.
Gram-positive bacteria are bacteria that give a positive result
in the Gram stain test. Gram-positive bacteria
take up the crystal violet stain used in the test, and then appear to be purple-coloured
when seen through a microscope. It also has thick layer of peptidoglycan in
cell wall and less lipids. The peptidoglycan traps the crystal violet in the
cytoplasm. The violet stain also is not washable even by using alcohol. The
example of gram-positive bacteria are Staphylococcus sp. and Bacillus sp.
In the experiment,
centrifugation method played the main role and did used for few times. A centrifuge is a
laboratory device that is used for the separation of fluids, gas or liquid,
based on density. Separation is achieved by spinning a vessel containing
material at high speed; the centrifugal force pushes heavier materials to the
outside of the vessel.
Objective
·
To
extract DNA from different organisms.
·
To determine
the purity of DNA.
·
To
determine the best ratio of purity.
·
To
differentiate DNA of gram-positive bacteria and gram-negative bacteria.
Procedure:
1. Centrifugation
1 ml of bacteria culture
grown to log phase was turned to pellet by centrifugation at 6000 x g for 2 min
at room temperature. The supernatant was decanted completely.
2. Resuspension of pellet
100µl Buffer R1 was added
to the pellet and the cells were resuspended completely by pipetting up and
down.
3. Lysozyme treatment
For Gram-negative bacteria
strains, 10 µl lysozyme (50mg/ml) was added into the cell suspension. For
Gram-positive bacteria strains, 20 µl lysozyme (50mg/ml) was added into
the cell suspension. They were mixed thoroughly and incubated at 37°C for 20
min.
4. Centrifugation
Digested cells were
centrifuged at 10000 x g for 3 min and pellet was formed. The supernatant was
decanted completely.
5. Protein denaturation
Pellet was resuspended in
180 µl of Buffer R2 and 20 µl of Proteinase K was added. It was mixed
thoroughly. Then, it was incubated at 65 °c for 20 min with occasional
mixing every 5 min.
6. Homogenization
400 µl without RNase A
treatment of Buffer BG was added and mixed thoroughly by inverting
tube several times until a homogeneous solution was obtained. It was incubated
for 10 min at 65°C.
7. Addition of Ethanol
200 µl of absolute
ethanol was added. It was mixed immediately and thoroughly.
8. Loading to column
The sample was transferred into a column assembled in a
clean collection tube.it was centrifuged at 10000 x g for 1
min. Flow through was discarded.
9. Column washing
The column was washed with
750 µl of Wash Buffer and was centrifuged at 10000 x g for 1 min. Flow
through was discarded.
10. Column drying
The column was centrifuged
at 10000 x g for 1 min to remove residual ethanol.
11. DNA elution
The column was placed into
a clean microcentrifuge tube. 100 µl of preheated Elution Buffer,TE buffer
was added directly onto column membrane and was allowed to stand for 2 min. It
was centrifuged at 10000 x g for 1 min to elute DNA.
Reminder:
1. All steps are to be
carried out at room temperature.
2. Wash Buffer
(concentrate) has to be diluted with absolute ethanol before use.
3. If precipitate forms in
Buffer BG, incubate at 55°c -65°c with occasional mixing until completely
dissolved
Results
|
Bacteria
|
260
|
280
|
Ratio=260/280
|
|
Lactobacillus
|
0.273
|
0.190
|
1.437
|
|
Lactobacillus
|
0.151
|
0.127
|
1.189
|
|
Salmonella
|
0.318
|
0.210
|
1.514
|
Discussion
Gram-negative cell is
bacterial cell having wall composed of a thin peptidoglycan layer of their cell
wall It consists of a triple layer and outermost
layer contains lipopolysaccharide (LPS). Salmonella is gram-negative cell.
Gram-positive cell is
bacterial cell having a thick wall of peptidoglycan layer and it can retain the
crystal violet stain in the gram staining method. Lactobacillus used in this
experiment are Gram-positive cell.
Lysozyme
is enzyme that break down bacterial cell walls
to improve protein or nucleic acid extraction efficiency. The enzyme acts by
catalyzing the hydrolysis of 1,4-beta-linkages
between N-acetylmuramic acid and N-acetyl-D-glucosamine residues in a peptidoglycan and between N-acetyl-D-glucosamine residues in chitodextrins.
Ethanol precipitation is
used to purify DNA and polysaccharides from aqueous solution by adding ethanol
as an anti-solvent. Since the DNA insoluble in ethanol but soluble in water, so
the ethanol can removes some of the salts present in the leftover supernatant.
The purity of DNA can be calculated by formula below:
Purity of DNA = OD260 / OD280
Ratio
between the readings at 260 nm and 280 nm (OD260/OD280) provides an estimate of
the purity of the sample. The best ratio of the purity is between 1.7-1.9.
Based on the result observed, the ratio of the purity is not lie in the
range between 1.7 and 1.9. This is due to several factors as below:
|
Factors
|
Possibilities
|
|
Incomplete cell suspension
|
Inefficient of cell suspension during the
experiment carried out.
|
|
Low elution efficiency
|
Elution buffer does not dispensed directly onto
the center of the membrane.
|
|
Contamination
|
Supernatant removed incompletely and still stick
with the fluid.
|
Conclusion
DNA
extraction of Gram-negative bacteria is much harder than DNA extraction of
Gram-positive bacteria. Precaution steps should be conducted to get the pure
DNA.
Reference
http://www.highveld.com/microbiolog
