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In blood separations, blood samples were drawn from the elected student
(Ella and Esperanza) at 8-9 mL by using a syringe. All samples were stored in the
vacutainer with anti-coagulant (K3-EDTA) and empty vacutainer. From each of the
sample, one mL of whole blood was transferred into a separated labeled vial. After
that, all of the vacutainer would be centrifuged at 3000g using Allegra x15 centrifuge
on 20C, for 10 minutes. Then, the plasma (transparent liquid which were present in
the upper layer of a vial coated with K3-EDTA) would be transferred into a new vial
and stored at -80C
SCC buffer 1X 0,8 ml was added to blood samples (prepared by assistant),
mixed and centrifuged (1 minute, 12000rpm in microcentrifuge). One ml of
supernatant was removed. One ml of 1X SCC Buffer was added, vortexed, and
centrifuged (1 minute, 12000rpm). Supernatant was removed. NaOAc 0,2 M 375 l
was added to each pellet, vortexed, then 10%SDS 25 l and Proteinase K (10mg/ml
H2O) 5 l were added, vortexed, and incubated (1 hour, 55C).
Phenol/choloroform/isoamyl alcohol 120 l was added, vortexed (30 seconds), and
centrifuged (2 minutes, 12000rpm). Aqueous layer was removed to 1,5 ml
microcentrifuge tube, cold 100% ethanol 1 ml was added, mixed, and centrifuged (2
minutes, 12000rpm). Supernatant was removed. TE Buffer 1X 180 l was added,
vortexed, NaCOOH 20 l 2M was added, mixed, cold 100% ethanol 500 l was
added, mixed and centrifuged (1 minute, 12000rpm). Supernatant was removed,
pellet was rinsed with 1 ml of ethanol 70% and centrifuged (1 minute, 12000rpm).
Supernatant was removed, pellet was air dried till dry and resuspended (1X TE
Buffer 200 l was added, incubated [55C, 30 minutes], vortexed), and then stored at
-20C.
Spectrophotometer BIORAD was used to determine the quantitation of DNA
concentration. Firstly, dilute the DNA sample (1:2) and filled the cuvette with 50 L of
1X TE buffer. Then, set the cuvette on the cuvette holder of spectrophotometer. In
this experiment, we would use double strand DNA as a type of nucleic acid. The
conversion factor will be determined to calculate the nucleic acid concentrations from
absorbance data and the spectrophotometer with 1X TE buffer was blanked. The
cuvette inserted along with DNA sample to read the absorbance (50 L final volume).
Then, the results of Absorbance 230,260, and 280 were recorded.
Each DNA sample 5l was loaded into agarose gel well (prepared by
assistant). DNA marker 6l was loaded as well. Lid was closed and electricity was
run from (-) end to (+) end (100V, 6W, 0.6A, 30minutes). Gel was taken out and
washed. Result was recorded using Versa Doc.
PCR reaction mixture of total 25 l containing 12.875 l dH2O, 5l 1X Buffer
PCR, 2l dNTP mix 200M, 1.5l MgCl2 1.5mM, 0.125l of Taq DNA Polymerase,
0.75l Forward Primer 0.3M, 0.75l Reverse Primer 0.3M, 2l DNA template for
each sample was set up in PCR tube. It was then put in PCR machine that has been
programmed (Predenaturation : 95C for 10 minutes, Denaturation : 94C for 30
seconds, Annealing : 62C for 30 seconds, Extension : 72C for 30 seconds, set for
35 cycles). Product was kept at 4C.
Cromas Lite was installed, DNA file was opened, edited and copied. BLAST
website was opened, then Blast Menu, Nucleotide Blast Menu were clicked
respectively. DNA Sequence was paste into Enter Query Sequence Area, Human
Genomic Database was chosen in search set. BLAST was run.
III. RESULTS
A. Blood seperation
Without anticoagulan
Figure 2. Agarose gel electrophoresis of DNA isolated from whole blood samples
C. Quantitation of DNA Concentration
DNA Concentration
1. Ellas Sample
A260 = (0.747 + 0.741) / 2 = 0.744
Concentration = (OD : ) x dilution factor
= (0.744 : 20) x 2
= 74.4 g/ml.
2. Esperanzas Sample
A260 = (0.858 + 0.838) / 2 = 0.848
Concentration = (OD : ) x dilution factor
= (0.848 : 20) x 2
= 84.8 g/ml.
DNA Purity
1. Ellas Sample
A230 = (0.292 + 0.291) / 2 = 0.2915
A260 = (0.747 + 0.741) / 2 = 0.744
A280 = (0.423 + 0.419) / 2 = 0.421
A260/A230 = 0.744 : 0.2915
= 2.55
A260/A280 = 0.744 : 0.421
= 1.77
2. Esperanzas Sample
A230 = (0.323 + 0.311) / 2 = 0.317
A260 = (0.858 + 0.838) / 2 = 0.848
A280 = (0.489 + 0.475) / 2 = 0.482
A260/A230 = 0.848 : 0.317
= 2.67
A260/A280 = 0.848 : 0.482
= 1.76
D. PCR
Figure 3 The PCR product of agarose gel electrophresis
E. DNA Sequencing
MDM2
IV. DISCUSSION
From the blood seperation result, blood which placed in EDTA vials, an
anticoagulan factor, clearly visible seperates into three layers. The first yellowish
transparant liquid layers is plasma, followed by thin layers called buffy coat which
consisted of leukocytes and platelets. And the third layer is whole blood consisted of
erythrocytes. This result was corresponding with the theory of classification of blood.
Those 3 layers were visible because centrifugation separates mixture based on its
weight. The seperated blood in no EDTA vials shows only two layers. The upper
layers is serum because there was a part of plasma called fibrinogen, which
combined with platelets, part of buffy coat, caused the blood to be coagulated. And
bellow is whole blood consisted of erythrocytes, leukocytes, and platelets. So, this
experiment was successfully done and supported the stated theory in Sherwood
(2010).
DNA was isolated from leukocytes because leukocytes have nuclei where
DNA is located. DNA isolation procedures was using whole blood because even the
blood was already separated into layers, leukocytes in buffy coat was too little to be
extracted. To get DNA only, other components in the whole blood must be removed
first. Unwanted protein could be removed by using proteinase K by degrading and
denaturating the proteins. After that using SDS detergent, cell membrane was lysed
so DNA in the nuclei could be extracted. PCl was then added to remove most of non
nucleic acid molecules and unwanted components. DNA then have to be precipitated
using Ethanol and resuspended using TE buffer. The result in this experiment was
whitish color pellet DNA in vial beacause the sample was really clean.
Electrophoresis was done to know the length of the sequences. Human
genome have more than 10.000 base pairs. In figure 2, it shows that our DNA
samples are slightly above the band formed by the DNA ladder. The first band in the
DNA ladder indicates the size of 10.000 base pairs. So, our DNA samples had more
than 10.000 base pairs. The unclear thin band was also appeared which meant that
the concentration of DNA of the sample was low. Lane 6, which was filled with
Esperanzas DNA was thicker compared to lane filled with Ellas DNA. These results
proved that higher concentration of Esperanzas DNA caused her DNA band was
thicker when it was seen in electrophoresis.
Quantitation of DNA concentration showed significant calculation for DNA
purity. DNA is considered pure if the purity index is between 1.8 2. Result below
1.8 indicates that the DNA sample was contaminated with protein and result above 2
indicates that the DNA sample was contaminated with RNA. Our DNA sample had
purity value 1.77 for Ellas DNA and 1.76 for Esperanzas DNA. Both purity index of
DNA sample calculation results was below 1.8 which meant that both samples were
contaminated with protein. Consentration of Esperanzas DNA (84.8 g/ml ) was
higher than Ellas DNA (74.4 g/ml ). This could happen due to error during pipetting
of DNA.
After the process of PCR, electrophoresis was performed once again to
confirm that we had amplified the right target DNA. Then, the sample was compared
with the DNA ladder to measure the size using Versa Doc Machine. Our gene target
was MDM2 with the size of 622 base pairs. Unfortunatelly in our result, no samples
DNA were shown. This could be due to error in PCR process or making the mix of
PCR reaction. Some materials might be forgotten to be added or not perfectly mixed
so couldnt be read by electrophoresis.
Lastly, BLAST result was to show that the amplified DNA sequence was really
MDM2 gene that we wanted. BLAST is used to compare our DNA sample with the
database. Identification of gene by BLAST was based on the local similarity between
the sequences. By using these tools, the sample of gene was 84% identified as
MDM2 gene.
Conclusion from our experiments, although there was some error we were
succeed to reach the goal of all of these experiments.
V. REFERENCE
Marks DB, Marks AD, Smith CM. Basic Medical Biochemistry : A Clinical Approach.
3rd ed. Lippincott Wiliams and Wilkins : Philadelphia; 2008
Cantor CR, Smith CL. Genomics: the science and technology behind the Human
Genome Project. John Wiley & Sons: Canada ; 1999.