IV.

DISCUSSION

We got two result from the blood separation experiment. The first result is we
can see there are three layers in the vacutainer with anti coagulant (ETDA). The
layers are plasma, buffy coat and red blood cell. Plasma is the transparent layers on
the top. The plasma are containing water, hormone, protein, nutrients, vitamin, lipid,
mineral, and etc. The second layer is Buffy coat. It is contain white blood cells and
platelets. The buffy coat is a thin white colour layer. The third layer is red blood cell.
It is red dark colour layer and containing hemoglobins. The second result is we can
see two layers in the vacutainer without anti coagulant. The layers are serum and
red blood cells. The serum are containing water, protein, white blood cell, and other.
The second layer is red blood cell. The blood is clotting and contains red blood cell,
white blood cell and platelet that have been coagulated. Our result is similar with the
references in the introduction part ( Vox Sang. 1999, NCBI)

In DNA isolation experiment we use the previous sample in blood separation,

The blood cells we used to get DNA were white blood cells, because they contained
nucleus. First, proteinase K was added to degrade the protein. SDS detergent was
then added to lyse the cell membrane, PCI to remove most of non-nucleic acid
molecules. Ethanol and TE buffer were lastly added to precipitate the DNA. After
DNA isolated, we check the that the DNA was succeed to be isolated with
electrophoresis. The electrophoresis will show The distance that DNA’s fragment
traveled in the gel indicates the length of DNA. Larger DNA strands travel shorter
distances and short strands travel longer distances. Our result is successful. Both
boy DNA and jen DNA have more than 10000 base pairs. Their DNA is in the form a
white dot in the vial and its like thin yarn. But in our experiment there are some
different in the thickness of the band . it is maybe occur because human error during
the experiment procedure (Lee, P. Y., Costumbrado 2012)

The DNA concentration was determined by the purity index range. DNA purity
can be achieved by calculating the value of A260 divided by A280 .Both of our DNA
is not pure. It was supposed to be between 1,8 – 2,0. If below 1,8, the DNA is
contaminated by protein. If higher than 2,0, the DNA is contaminated by RNA. Our
DNA result show that purity index DNA Boy and Jen (A260/A280) are not pure.
Because our purity sample is 1,5. It is maybe because our DNA is contaminated by
protein. Our DNA purity index (A260/A230) is also not pure. The purity is -1,275 and -
0,650. Both Jen and boy DNA is contaminated by the phenol,etc. The conclusion is
our experiment is not good.(Carlos F. Barbas III, et al. 2001)

We do another electrophoresis was performed to ensure we amplify the right

DNA sequence. Lane A was the DNA marker, lane B and C are Boy’s and Jenifer’s
samples, lane D is control positive . If our experiment is done correctly, there should
be a band formed in the lane D (positive control). My sub unit samples are faint and
didn’t show up. it might be because of NaOAc is volatile and easy to evaporate or
The error most likely maybe occurred during the pipetting the cofactor. The other sub
unit got it right. Their Sample shows the band in lane B and C. Their control positive
show the band at about 800-900 base pairs. But there are different thickness about
sample in Lane B 1 (Boy) and sample 2 in Lane C (jenifer). The sample in lane c is
have thick band. It is might be due to excessive usage of cofactor (MgCl2). (Elizabeth
van Pelt-Verkuil , et al , 2008)

In DNA sequencing , we use BLAST to comparing our sample with the

database. BLAST is shown us that our amplified DNA is similar with TGFBR-1 gene.
From the result of the comparation, we can conclude that the gene that was
amplified was really TGFBR-1 gene (99% similarity). It is means that our experiment
is successful. (Clyde A. Hutchison, II Nucleic Acids Res. 2007 Sep,NCBI)
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