2
1.8
Concentration (ug/ml)
1.6
1.4
1.2
1
0.8
0.6
0.4
0.2
0
Root
Stem
Leaf
Flower
Samples
Figure 1. The Protein Concentration in Each Sample. The concentration was determined
through the use of Bradford Assay. The standard curve of a known protein obtained at
595 nm was used (fig. 2). The equation X=(Y+0.0045)/0.0175 of the fit line from the
standard curve and the absorbance at 595 nm of different amounts of samples (average)
(table 1b) was used to calculate average concentration of proteins in samples.
Amount of Sample Used in Water to total 3 ml
5 ul
10 ul
20 ul
r
0.055
0.102
0.169
s
0.004
0.017
0.038
l
0.01
0.026
0.048
f
0.032
0.082
0.159
Table 1a. The Absorbance at 595 nm of Different Amounts of Protein samples.
Samples
Average protein concentration
1.78428571ug/ml
0.34142857ug/ml
0.49ug/ml
1.37857143ug/ml
Table 1b. Average concentration of protein from each sample calculated from
absorbances (tab 1a) and the standard curve (fig 2).
Absorbance (OD)
Concentration of Protein vs. Absorbance at 595 nm
0.18
0.16
0.14
0.12
0.1
0.08
0.06
0.04
0.02
0
y = 0.0175x - 0.0045
2
R = 0.9866
0
2
4
6
8
10
Concentration (ug/ml)
Figure 2. The standard curve used to calculate the amount of protein in each sample.
The absorbance of known concentration of protein with Comassie Brilliant Blue G-250
measured at 595 nm was used to make this curve.
Samples
Flower
Leaf
Stem
Root
Absorbance at
wavelengths:
260 nm
280 nm
DNA to
Protein
ratio
0.463
0.239
1.937
0.406
0.212
1.915
0.188
0.097
1.938
0.324
0.185
1.751
Table 2. The Extracted DNA Absorbance at 260 and 280 nm. The original DNA extract
was diluted to 5.0E-3 to obtain an absorbance at 0.1-1. The purity of the DNA extracted is
measured by dividing the absorbance at A260 by A280. A value at a range of 1.8-2.0
indicates pure DNA.
A-50 ug
L
R
F
S
Sample
A-100 ug
B-50
R
F
L
S
S
R
F
L
B-100
S
R
L
F
Table 3. The order from highest to lowest DNA content (lowest Ct value to highest Ct
value) in F-flower, L-leaf, S-stem, R-root of the mum determined by the use of two
primers (A, B) and two different amount of DNA (50 and 100-ug). Note that Primer A
amplified the right gene and the 100-ug samples were used to determine results.
Results
Referring to figure 1, the roots of the plant had the highest concentration of protein per
ug followed by the flower, leaf and stem. The ratios of absorbance of DNA to the
absorbance of protein in the DNA extracted from the different parts of the plant are
shown in table 2. The flower, leaf and the stem are between 1.-2.0 but the root is 1.75, but
we can still assume that all sample had pure DNA.
The RT-PCR data didn’t give a coherent result. Two different amounts of DNA were
used for each sample in each primer. One sample was 50 ug and the other was double the
first amount (100 ug). Primers A and B amplified different length of DNA; a 500bp and
1400bp consecutively. As shown in the gel figure, primer B amplified a band of 800 bp
and one band of that seems to be of ~1400 bp in the leaf 50ug sample. Primer A amplify
a 500 bp fragment as expected. As shown in the melting curve and the agarose gel, there
are lots of impurities in the sample…..
Discussion:
Reason for choosing the plant:
The mum was chosen because it is commonly used for decoration. People request more
variations of them. It would make the job easier for the scientists who are making
variation to the plant to know as much as they can about the plant. It is also very
important to now where the most amount of the rubsco coding gene is incase someone
wanted to isolate the code for some other use. (may be even find more rubisco in that
area). This knowledge could also be used to know
The amount of Rubisco in the flower, the leaf, the stem and the root was measured
indirectly using total protein concentration in the samples and the amplified DNA region
of chloroplast that codes for Rubisco protein. It was found that all the samples contain
chloroplasts or probably some other kind of plasts.
The results of the RT-PCR reaction show that the roots of the mum have more rubisco
followed by the leaf, stem and flower. The leaves do not carry as much rubisco as the
roots do, as shown in Table 3. According TO OUR SHAKY RESULTS Our hypothesis,
THAT THE LEAVES WOULD HAVE MORE RUBISCO, is rejected surprisingly.
The PCR reaction that were performed didn’t all have the expected results. For one,
primer B didn’t amplify at the right fragment size. After the amplification a sample was
run on agorose gel to check for a 1400 bp fragment that the primer is supposed to amplify
(fig.. ). The primer B lane had a 800bp fragment. This leaves us with Prime A as our
champion. We chose to use the 100 ug samples to form a conclusion because primer A
doesn’t work as well in a 50 ug sample and because the 100 ug sample is easier to pipet
and so there might be less pipeting errors in those samples. To make the result of the
experiment more conclusive and stronger one should use a different primer rather then
prime B.
The set of PCR reaction with primer A and 100ug of DNA shows that the there are more
copies of the rubsco gene in the roots. This could be because the roots use more energy;
so there would be more chloroplasts, which are carring the rubisco gene. (research).
In second place for amount of rubsco gene possession is the leaf. (list the reasons).
In third place for rubisco possession is the stem (list reasons). And in last place is the
flower ( so sad).
The protein concentration results (fig.1) shows that the roots have the most total protein
followed by the flower, the leaf and the stem. (The total protein concentration tests
determined that the most amount of protein is located in the roots.)
Find out why
The roots make more protein because they need to grow to provide nutrients for the
plants. And the roots contain more plastids which each have their own copy of the
rubisco gene.
Function of the roots:
In second place for protein possession are the flower petals, which according to the PCR
reaction have the least amount of rubsco genes. The flower is involved in reproduction
(list reasons, research).
In third place comes the leaves ( suprising since leafs do blah blah Research). Finaly the
stem has the least amount of total protein concentration per ml (for reasons such as
(research).
The western blot (fig ) wasn’t successful in identifying all rubisco protein in all the
samples. But we can see on band in it, so I guess we did the procedure ok. The wester
blot didn’t show the relative amount of Rubisco Protein in each sample. Although if it did
we would guess with 10 % certainty that the results would match the PCR reactions.
The western blot might have not worked well becaue the amount of protein set in each
well wasn’t equal, or probably the usual pipeting error affected it. Or quit possibily, the
absence of bands might just mean that there was rubisco protein in those samples, and if
there was it was too little to be detected by the method we used. To accqure a better result
we should repeat the experiment and try to add reverse RNA transcription to get a more
clear comparison of rubisco gene expression in each part of the plant.
To further the experiment one would try to find out more accurately why there would be
more expression in the roots. One would change the amount rubisco in that area and see
the concequences, and thus find the function of the rubisco in that area.
Or the result could suggest that we have made a drastic mistake in pipeting or calculating
concentrations
Leaf
Leaves
Leafs