LAB 6: BACTERIAL DNA EXTRACTION
SCHOOL OF TECHNOLOGY
INDUSTRY
UNIVERSITY SCIENCES
MALAYSIA
DEGREE IN BIOPROCESS
IBG 102
BIOLOGY FOR
TECHNOLOGIST
Name
|
1. LOH SHI WEI (137602)
2. LAI CHONG SING (137592)
3. SITI NORASYIKIN BINTI SALMI (137673)
4. SITI NUR SUHAILI AFIQAH BINTI SARIMAN
(137674)
5. NUR LIYANA ATHILAH BINTI MOHD AFFANDI
(137636)
|
Title
|
LAB 6: BACTERIAL DNA
EXTRACTION
|
Date
of practical
|
24/10/17
|
Date of Report Submitted
|
07/11/17
|
Lecturer
|
DR MOHD NAZRI ISMAIL
|
LAB
6: BACTERIAL DNA EXTRACTION
INTRODUCTION
DNA
isolation is a process of purification of DNA from sample using a combination
of physical and chemical methods. With a pure sample of DNA, we can test a
newborn for a genetic disease, analyse forensic evidence, or study a gene
involved in cancer.
Plasmid
is a small DNA molecule within a cell that is physically separated from a
chromosomal DNA which can replicate independently. Plasmid are commonly found
in bacteria as small circular, double stranded DNA molecule.
Plasmid
are involved in the DNA recombinant experiment to produce large quantity of
DNA. Plasmid act as vector to clone specific gene in DNA. They are often found
in Gram-positive bacteria and Gram-negative bacteria.
Extraction
of plasmids DNA is a classical method of isolating plasmid DNA. The name of the
method is Alkaline Lysis. By going through the steps within this experiment, it
will be quicker and easier to yield plasmid DNA from any bacteria. This method
isolated plasmid DNA or other cell components such as proteins by breaking the
cell open. Bacteria containing the plasmid of interest is first grown, and then
allowed to lyse with an alkaline lysis buffer consisting of a detergent sodium
dodecyl sulfate (SDS) and a strong base sodium hydroxide.
In
general, plasmids are not essential for the survival of bacteria, but they
nevertheless encode with a wide variety of genetic determinants, which permit
their bacterial hosts to survive better in an adverse environment of to
complete better with other microorganisms occupying the same ecological niche.
OBJECTIVE
To
study the method for bacterial DNA extraction
MATERIALS
AND APPARATUS
Bacteria culture
100 micro liter Buffer
R1
20 micro-liter lysozyme
( gram positive bacteria)
100 micro-liter Buffer
R2
20 micro-liter
Proteinase K microorlit
20 micro-liter RNase
440 micro liter Buffer
BG
200 micro-liter
absolute ethanol
650 micro-liter Wash
Buffer
50 micro-liter Elution
Buffer
Centrifuge
Pipette
Column
Collection tube
Microcentrifuge tube
Beaker
Incubator
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.
Picture
1: sample was put into centrifuge
Picture
2: setting of centrifuge at 6000xg for 2 minutes.
2.
Resuspension of Pellet
100µl Buffer R1 was
added to the pellet and the cells were resuspended completely by pipetting up
and down.
Picture 3: addition of Buffer R1 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.
Picture
4: the final result obtained from the procedure before checking the absorbance.
12. ABSORBANCE READING
result from step 11 was used to read the absorbance at 260nm and 280nm.
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.
RESULT:
OD 260
|
OD280
|
Ratio
|
|
Blank
|
0.053
|
0.048
|
-
|
Lactobacillus A
|
0.099
|
0.07
|
2.091
|
Lactobacillus B
|
0.094
|
0.071
|
1.783
|
Purity of sample :
Type
|
Lactobacillus
A
|
Lactobacillus
B :
|
Purity
|
(0.099-0.053)
/ (0.07-0.048) = 2.1
·
Not pure
|
(0.094-0.053)
/ (0.071-0.048) = 1.8
·
Pure
|
Concentration of bacteria
|
OD
260 ;
(0.099-0.053)
/ (0.02*0.051) = 45.098 μ/mL
OD
280 ;
(0.07-0.046)
/ (0.02*0.051) = 21.57μ/mL
|
OD260;
(0.094-0.053)
/ (0.02*0.051) = 40.20 μ/mL
OD
280;
(0.071-0.048)
/ (0.02*0.051) = 22.55 μ/mL
|
DISCUSSION
This
experiment is conducted to study step for DNA extraction. The bacteria used in
this experiment is Lactobacillus sp.
The experiment conducted in a duplicate in order to obtain accurate result.
This bacteria is gram positive bacteria hence, 20 µl
lysozymes
is added. Lysozymes is added to break
down the bacterial cell walls. The enzyme damages bacterial cell
walls by catalyzing hydrolysis of 1, 4-beta-linkages
between N-acetylmuramic acid and N-acetyl-D-glucosamine residues
in a peptidoglycan. The ratio of absorbance at 260nm and 280nm (OD260/OD280) is
use to assess the purity of DNA and RNA. A ratio of 1.8 is generally accepted
as pure for DNA and a ratio of 2.0 is generally accepted as pure for RNA. If
the ratio is lower in either case, it may due to presence of protein, phenol or
other contaminant that absorb strongly at or near 280nm. From the result obtained, the ratio for Lactobacillus A is 2.1 and for Lactobacillus
B is 1.8. from the result can be conclude that sample for Lactobacillus A
was contaminated as the ratio is higher than 1.8 this is may due to residual
RNA from nucleic acid extraction. Result for Lactobacillus B can be concluded
as pure as the ratio is 1.8.
CONCLUSION
In
conclusion, the objective of the experiment to study the method for bacterial
DNA extraction is success even though the result obtained shows that the sample
was contaminated during the process of DNA extraction. Precaution step need to
be taken in order to have a good result without contamination.
REFERENCES
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