Kevin Reynolds
School of Natural Sciences at Ferrum College
kreynolds@ferrum.edu
I. Abstract
Genomic research is the study of an organism’s genome. This information can be used to
find out valuable information about an organism’s genetic makeup, including determining the
specific functions of specific genes within an organism. For the following procedure, my
colleagues and I extracted genomic DNA from Solenostemon scutellarioides, cloned it, and
eventually had it sequenced. We focused on GAPDH, the housekeeping gene for many
organisms. It was found that while the gene we sequenced is common to many organisms,
the one we were interested in has yet to be sequenced.
II. Introduction
Genomic research is the study of the role that genes have in individual organisms and
attempts to sequence part if not all of an organism’s genome. Whether it is to find
differences amongst ethnicities, which genes cause specific diseases, what roles certain
genes play, or to sequence a genome, genomic research is a rapidly growing field (5).
Cloning is a large part of genomic research. Cloning is replicating DNA, whether it is a piece
of DNA or an entire organism. There are many reasons to use cloning, which include
medical purposes, revive endangered animals, etc. The three different types of cloning are
DNA cloning, reproductive cloning, and therapeutic cloning. Specifically, I will be doing
DNA cloning, which is the transfer of DNA from one organism to a self-replicating bacterial
plasmid (6).
For this project, I cloned and sequenced the housekeeping gene from the Coleus plant,
Solenostemon scutellarioides. The housekeeping gene, for an organism, is a gene that is
generally always expresses, as it codes for proteins that are vital for basic cellular function.
In this case, GAPDH, or Glyceraldehyde-3-phoshate dehydrogenase is the housekeeping
gene. GAPDH is an acidic protein and is an important enzyme in the processes of glycolysis
and gluconeogenesis, which occurs in the cytoplasm. GAPDH is a tetramer, having four
subunits, which are independent of each other and contain NAD+. GAPDH is one of the
most commonly used housekeeping genes used in comparison of gene expression. It is
used to catalyze the conversion of glyceraldehydes 3-phosphate to 1,3-diphosphoglycerate
by converting NAD+ to NADH. It has also been found recently that GAPDH is a
multifunctional protein that is involved in many subcellular processes, mainly in critical
nuclear pathways (1).
Solenostemon scutellarioides, or the Coleus plant, is a herbaceous perennial. It is
commonly found from Malaysia to southeastern Asia. It can grow up to three feet tall and
three feet in diameter. It’s flower color ranges from blue to white. It has been popular
since Victorian times, and has been one of the most hybridized plants over the years (3).
For this study I used current molecular techniques as well as bioinformatics to clone
and sequence the housekeeping gene, GAPDH, from Solenostemon scutellarioides. I spent
about ten weeks using current technology and methods to complete this study. Using a
broad range of technology and methods including PCR, DNA purification, and gel
electrophoresis to obtain a valid gene sequence for my gene of interest. As there are
multiple GAPDH genes, I will be specifically targeting the GAPC genes(4).
III. Methods
a. Extracting Genomic DNA
I first extracted the genomic DNA or gDNA from my plant of choice, Tradescantia
pallida, also known as Purple Heart. Isolating the genomic DNA is the first step for many
procedures such as cloning. During this step, I separated the DNA from proteins and other
components that are inside the cells. This process needs to be done with care to ensure that it
remains intact during the purification process. It is also necessary to purify the gDNA and rid it
of any enzymes and other acidic contents, which can cause complications further on in the
procedure.
I first collected a 100mg leaf sample of Tradescantia pallida. I then cut it into 1mm
pieces, with a new/clean razor blade, and placed it into a 1.5ml microcentrifuge tube with 200ul
of lysis buffer. I used a clean micropestle to grind the plant material for three minutes, until the
plant material was ground to very fine particles. An additional 500ul of lysis buffer was then
added to the microcentrifuge tube, and I continued to grind the plant matter until I had a
homogenous mixture. The microcentrifuge tube was then microcentrifuged for five minutes at
room temperature at 12,000rpm. Once my sample was done, I took 400ul of supernatant, being
careful not to disturb the pellet, and placed it into another microcentrifuge tube with 500ul of
70% ethanol. The ethanol and supernatant was mixed using a pipet. 800ul of the lysate and
ethanol mixture was transferred to a mini DNA extraction column, which was inside of a 2ml
capless collection tube. The column and collection tube were then placed in the microcentrifuge
for one minute at 12,000rpm at room temperature. The liquid in the collection tube was then
discarded. 700ul of wash buffer was added to the column, centrifuged for one minute, discarding
any flow through once done. This process was repeated two more times. After the final wash
the DNA extraction column was placed back in its capless collection tube and centrifuged for
two minutes to allow it to dry. The DNA extraction column was then transferred to a clean
microcentrifuge tube, and 80ul of 70°C sterile water was added to the column, making sure the
water was on the bed. After sitting for one minute, the column (still inside the microcentrifuge
tube) was centrifuged at 12,000rpm for two minutes. The column was removed and discarded,
while the microcentrifuge tube was capped and stored at -20°C.
b. GAPDH PCR/Initial PCR
PCR, or polymerase chain reaction, is a technique used to rapidly create multiple copies
of a segment of DNA utilizing repeating cycles of DNA synthesis. This technique allows small
quantities of DNA to be amplified into large amounts, which can be used for further
experimentation. This is how the small amount of gDNA I collected will be replicated into
quantities large enough to be useful.
I planned my first round of PCR. I performed PCR on my plant gDNA, control plant
gDNA and pGAP plasmid DNA as two positive controls, and sterile water as a negative control.
The table below shows how I planned my PCR reactions:
Tube Label
Template
1-gDNA
gDNA I collected
2-control gDNA
Control DNA
3-pGAP
pGAP plasmid DNA
4-H2O
Sterile water
A Master Mix, Bio-Rad 2X master mix, was obtained containing, which contained Taq
DNA polymerase, dNTPs, buffer, and salts. Primers were then added to form a complete 2X
master mix with initial primers-2X MMIP. 20ul of 2X MMIP was needed for each reaction.
The initial primers were supplied at a 100uM and the concentration of primers in the 2X MMIP
was 2uM. The 2X MMIP, once prepared, was mixed with a pipet and stored on ice. Following
my table for the PCR, I labeled my PCR tubes and placed each of them into a tube adaptor and
capped them. Each PCR was set up with the following reagents:
Reagent
Amount
2X MMIP
20ul
Sterile Water
15ul
DNA template or negative 5ul
control
Total
40ul
The reagents were mixed together using a pipet. The PCR tubes were then placed in a thermal
cycler using the following Initial GAPDH PCR program:
Initial denaturation:
95°C for 1 minute
Then 40 cycle of:
Denaturation:
95°C for 1 minute
Annealing:
52°C for 1 minute
Extension:
72°C for 2 minutes
Final Extension:
72°C for 6 minutes
Hold:
15°C
c. Nested PCR
Nested PCR is used to increase the yield and specify the amount of the targeted DNA. It
does this by using two sequential sets of primers. The first set binds to sequences outside the
DNA, while the second set bids to sequences in the target DNA that are within the portion
amplified by the first set. So, it gets the gDNA closer to what we are actually targeting.
I first planned my Nested PCR or Second-Round PCR. I used my plant gDNA, control
plant gDNA and pGAP plasmid DNA as two positive controls, and sterile water as a negative
control. The table below shows how I planned my PCR reactions:
Tube Label
Template
1-gDNA
gDNA I collected
2-control gDNA
Control DNA
3-pGAP
pGAP plasmid DNA
4-H2O
Sterile water
Next, I prepared my mater mix. 20ul of 2X master mix and yellow nested primers2XMMNP was required for each PCR reaction. The yellow nested primers were supplied at a
25uM concentration and the concentration of the 2XMMNP needed to be 0.5uM. This was
prepared no more than thirty minutes before use. It was mixed with a pipet and stored on ice. I
obtained my tube of gDNA from the initial round of PCR. I added 1ul of exonuclease I to my
initial PCR sample of amplified DNA. The tube was then incubated at 37°C for fifteen minutes
and then at 80°C for fifteen minutes, which inactivated the exonuclease I enzyme. I then diluted
the initial PCR sample by 100X. Using my Nested PCR plan, I set up my PCR reactions. The
following table shows how I set up my PCR reactions:
Reagents
Amount
Yellow master mix-2X MMNP
20ul
DNA template or negative control
20ul
Total
40ul
The reagents were mixed using a pipet once each reaction was prepared. The PCR
reactions were then placed in the thermal cycler using the following Nested GAPGH
PCR program:
Initial denaturation:
95°C for 5 minute
Then 30 cycle of:
Denaturation:
95°C for 1 minute
Annealing:
46°C for 1 minute
Extension:
72°C for 2 minutes
Final Extension:
Hold:
72°C for 6 minutes
15°C
d. Electrophoresis
Agarose gel electrophoresis separated DNA fragments based on their size. PCR products
are loaded into the agarose gel, which is in a chamber filled with conductive buffer solution.
Negatively charged DNA will be forced toward the positive pole. The distance the DNA
fragment travels through the gel is inversely proportion to the size of the fragment. This will
allow me to see if my PCR was successful, if anything was contaminated, and the number and
size of the PCR products.
I planned my gel electrophoresis using the following table:
Lane Number
Sample
8
800bp
Sample Volume(ul)
molecular 10
weight ruler
7
Initial PCR 4
20
6
Initial PCR 2
20
5
Initial PCR 1
20
4
Nested 4
5
3
Nested 3
5
2
Nested 2
5
1
Nested 1
5
NOTE: The molecular weight ruler contained band sizes of 500, 1000, 1500, 2000, 2500, 3000,
3500, 4000, 4500, 5000.
NOTE: The second positive control from the initial PCR was not used due to there not being
enough wells.
Next, I prepared my samples. 20ul of each initial PCR reaction was transferred to a
labeled microcentrifuge tube and 5ul of 5X loading dye was added to each tube and mixed
together with a pipet.
5ul of each yellow nested PCR was transferred into a labeled
moicrocentrifuge tube and mixed with 1ul of 5X loading dye. A 1% agarose gel was prepared,
the samples were loaded, according my plan, and the gel was run at 100V for thirty minutes.
Once complete, the gel was looked at and photographed.
e. GAPDH PCR/Initial PCR Attempt #2
It was necessary that this part of my procedure was performed again, reasons for which
will be discussed in the results section.
The same steps were used from part b-GADPH PCR, with a few changes. I only ran
three PCR reactions which included my gDNA sample, positive control (plasmid DNA), and
negative control(sterile water). Master mix was prepared for only three samples, using blue
primers. The rest of the procedure remained the same.
f. Electrophoresis on GAPDH PCR Attempt #2
The same procedure was used as it was for part d, Electrophoresis. The gel was loaded as
follows:
Lane Number
Sample
Volume (ul)
1
500bp molecular weight ruler
10
2
Initial PCR gDNA sample
20
3
Initial PCR control-plasmid DNA
20
4
Initial PCR sterile water
20
g. PCR Purification
Before the PCR product can be used for cloning, it must first be purified. PCR reation
mix contains dNTPs, primer-dimers, unused primers, and DNA polymerase, which must be taken
out, as they can interfere later on in the procedure. This will take several steps, as one method is
not sufficient to remove all contaminants.
I obtained the yellow nested PCR for the plant gene fragment to be cloned. I obtained a
PCR Kleen spin column and resuspended the beads by flicking them and returned them to the
bottom of the column with a sharp downward flick. I discarded the cap and bottom of the spin
column and placed the column in a capless collection tube. The capless collection tube and the
spin column were centrifuged for two minutes at 735 x g, not using the top speed. The spin
column was moved to a microcentrifuge tube and the flow through and collection tube were
discarded. 30ul of the yellow nested PCR reaction was placed on top of the beads in the spin
column and centrifuged for two minutes at 735 X g. The spin column was discarded and the
microcentrifuge tube was capped.
h. Ligation
Once a gene has been amplified using PCR, the next step is to insert the DNA into a
plasmid or cloning vector so the fragment can be propagated. Ligation is the process of joining
two pieces of linear DNA into a singly piece by using DNA ligase. DNA ligase catalyzes the
formation of a phosphodiester bond between the 3’ end of one piece of DNA and the 5’end of a
second piece of DNA.
Before I started, I briefly spun down the stock solution tubes of 2x ligation reaction and
proofreading polymerase to collect contents at the bottom of the tube. I set up a blunting
reaction with the following reagents:
Reagents
Amount (ul)
2X ligation reaction buffer
5
Purified PCR product
1
Sterile Water
2.5
Proofreading Polymerase
0.5
Total
9
All of the reagents went into a labeled microcentrifuge tube, mixed well, and capped. The tube
was breafly centrifuged and placed in a water bath at 70°C for five minutes. Once taken out, the
tube was cooled on ice for two minutes. Once cooled, the tube was briefly centrifuged. A
ligation reaction was setup using the following reagents:
Reagent
Amount (ul)
Blunt Reaction
9
T4 DNA ligase
0.5
pJet 1.2 blunted vector
0.5
Total
10
The tube was capped, mixed well, and centrifuged. The tube sat at room temperature for ten
minutes.
i. Transformation
Once a gene has been amplified using PCR and ligated into a plasmid, the next step is to
introduce the plasmid into living bacterial cells so that it can be replicated. In this case, I will be
using E. coli as the living cell, which will be used to clone the gene of interest.
Before the transformation process, I prepared competent cells. To do this I first pipetted
1.5ml of C-Growth Medium into a 15 ml culture tube, and warmed to 37°C for atleast ten
minutes until it was needed. Two LB Amp IPTG plates were obtained, one for the ligation and
one as a control, and placed in an incubator at 37°C. 150ul of fresh starter culture, inoculated the
previous day, was placed into the pre-warmed C-Growth Medium and placed in a 37°C water
bath shaking at 200-275 rpm for 20-40 minutes.
Transformation buffer was prepared by
combining 250ul of transformation reagent A and 250ul of transformation reagent B in a
microcentrifuge tube, mixed, and stored on ice. after the 20-40 minute incubation, the actively
growing culture of C-Growth Medium was transferred to a microcentrifuge tube to obtain
competent cells. I centrifuged the bacteria at top speed for one minute and immediately put the
tube on ice. Keeping the cells on ice, the supernatant was removed. I then, very gently,
resuspended the bacterial pellet with 300ul of ice-cold transformation buffer, and incubated it on
ice for five minutes. The bacteria was centrifuged for one minute at top speed and placed back
on ice immediately. The supernatant was then removed, avoiding the pellet. While on ice, the
bacterial pellet was resuspended, gently, with 120ul of ice-cold transformation buffer. The
competent cells were then incubated on ice for five minutes.
Two microcentrifuge tubes were obtained, one for pGAP transformation and one for
transformation of my plant gDNA transformation. 1ul of control pGAP plasmid was placed on
one tube, and 5ul of my ligation reaction from step h-ligation was placed in the other, and both
were kept on ice. The competent cells were resuspended, and 50ul were placed into the pGAP
transformation tube, gently mixed, and returned to the ice. The same was done to the gDNA
transformation tube. The transformations were incubated on ice for ten minutes. The LP AMP
IPTG agar plates were retrieved and the entire transformations were pipetted onto its
corresponding plate. The bacteria was quickly spread over the plate and placed upside down,
back in the incubator at 37°C overnight. The next day they were analyzed.
The transformed colonies were grown in liquid culture minipreps. I obtained
25ml of LB Amp broth. I also obtained four 15ml culture tubes and 5ml of the LB Amp broth
was placed into each tube. A sterile loop was used to transfer a single colony from the LB Amp
IPTG plate containing the plated bacteria transformed with my plant gene ligation reaction to all
four culture tubes. The miniprep cultures were allowed to grow overnight at 37°C in a shaking
water bath.
j. Plasmid Purification
Once the plasmid has been inserted into the competent bacterial cells ad the cells have
been grown into colonies on a medium selective for cells containing the plasmid, the next step is
to perform a miniprep of the plasmid DNA to prepare for DNA sequencing. In this step, I
removed the plasmid from the E. coli, which gave me the cloned gene of interest.
A capless collection tube, plasmid mini column, and two microcentrifuge tubes for each
miniprep culture, from part i-transformation, was obtained and labeled. Each column was placed
in its capless collection tube.
1.5ml of each miniprep culture was transferred into each
microcentrifuge tube. The microcentifuge tubes were centrifuge at top speed for one minute.
The supernatant was removed, avoiding the pellet. I resuspended the pellet using 250ul of
resuspension solution, which was done to each tube, making sure no clumps of bacteria remain.
250ul of lysis solution was added to each tube and each tube was inverted gently six to eight
times. Within five minutes of adding the lysis, I added 350ul of neutralization solution and a
precipitate formed.
Each tube was centrifuged for five minutes at top speed.
Avoiding
disturbing the precipitate, I decanted the supernatant into the appropriate column. The columns
in their capless tubes were microcentrifuged for one minute at top speed. The flow through was
discarded and the column was placed in the collection tube. 750ul of was buffer, with ethanol,
was added to each column.
The columns were spun for one minute at top speed in the
microcentrifuge, and flow through was discarded.
The column was placed back into the
collection tube and spun for one minute to dry the column. Each column was transferred to a
clean microcentrifuge tube, and 100ul of elution solution was added onto the column, which was
allowed to absorb for one to two minutes. The columns were centrifuged for two minutes at top
speed. Afterward, the tubes were capped and the columns were discarded.
The next step was Restriction Digest Analysis. I first prepared a 2x master mix for Bgl II
restriction digestion reactions in a microcentrifuge tube using the following reagents:
Reagents
Volume for 4 Reactions
10x Bgl II reaction buffer
8ul
Sterile Water
28ul
Bgl enzyme
4ul
Total
40ul
The digestion reactions were then prepared by combining 10ul of the Bgl II master mix and 10ul
of each plasmid DNA in microcentrifuge tubes. The reactions were then incubated at 37°C for
one hour.
I then ran a gel electrophoresis, the plan for which is shown below:
Lane Number
Sample
Volume (ul)
1
500bp molecular weight ruler
10
2
Undigested 1
20
3
Digested 1
20
4
Undigested 2
20
5
Digested 2
20
6
Undigested 3
20
7
Digested 3
20
8
Digested 4
20
For the undigested sample, 15ul of sterile water was combined with 5ul of undigested DNA. The
samples were briefly centrifuged, 5ul of 5x loading dye was added, and the samples were put
into a 1% agarose gel and run at 100V for thirty minutes. The gel was then analyzed to
determine the concentration of the plasmid.
k. DNA Sequencing
Once I have my cloned gene, it can now be sequenced. There are several ways a gene
can be sequenced, but they all give the same information, the exact order of nucleotide bases.
After the gel was analyzed, I carefully chose a sample and sent it, along with a calculated
amount of primer to a lab at Virginia Tech to be sequenced. Once I got the sequence back, I
analyzed all of the results using iFinch, DNA Baser, and NCBI to analyze the results.
IV. Results
After I extracted my gDNA from Tradescantia pallida, I used a spectrophotometer to
quantitate the gDNA I extracted. Readings were taken at wavelengths of 260nm and 280nm.
My results showed A260=0.131 and A280=0.078. The purity of my DNA was found to be
[(0.131)/(0.078)]= 1.67. The concentration was found using [A260 *50ng/ul * dilution factor] so
[0.131 *50ng/ul * 5= 32.75ng/ul. The purity of my DNA and the concentration indicate that I
have a usable DNA sample, and can continue with my procedure.
During the first GAPDH/Initial PCR, I was only able to do run my gDNA sample, gDNA
positive control, and the sterile water negative control due to space restrictions in the thermo
cycler. Later, it was also discovered that the thermo cycler had stopped at thirty-nine cycles, not
performing the final extension. At this point, I was undetermined whether or not PCR was
successful.
After Nested PCR, gel electrophoresis was performed on my samples. Picture 1.1 shows
the gel after it had been run.
Picture1.1
Lane:
1
2
3
4
5
6
7
8
The only bands that can be seen are the 500bp molecular weight marker, in lane 8, and the
Nested PCR pGAP, in lane 3. This indicates that the Nested PCR was successful, but the Initial
PCR was not successful, possibly due to the fact that it only did thirty-nine cycles and did not do
the final extension.
A new thermo cycler was purchased and used to perform Initial PCR again, since it did
not work the first time. There were not enough of the yellow primers for Nested PCR so only
Initial PCR was performed. Gel electrophoresis was done after the second round of Initial PCR,
the results of which can be seen in picture 2.1.
Picture 2.1
Lane: 1
2
3
4
By looking at lane 3, positive control, it is evident that the Initial PCR was successful this
time. However, I do not have a band in lane 2, which is my gDNA sample. This leads me to
believe that I was either unsuccessful in extracting gDNA or that something, I can not explain,
happened to my sample of gDNA. There was a slight band in lane 2 and 4, but this was most
likely due to lane 3 leaking, as lane 4 was a negative control.
At this point, I took a sample of kaffir lily,cilivia miniata, from Darryl Tarver. I used this
to proceed with my procedure.
During the transformation process, It was necessary to determine whether or not the
ligation and transformation process was successful, to know it I could proceed or not. Table 2.1
shows how many bacterial colonies grew on the LB Amp IPTG agar plates.
Table 2.1
Transformation
Number of Colonies
Control pGAP plasmid
Too Numerous to Count
Plant Gene Ligation
153
Based on the number of bacterial colonies that grew, the ligation and transformation were
successful.
After plasmid purification, gel electrophoresis was done to determine if I was successful
up to this point. Picture 3.1 shows the resulting gel.
Picture 3.1
Lane:
1
2
3
4
5
6
7
8
Although I had some bands present, I did not have two bands present for any of the digested, in
lanes 3,5,7, and 8. This leads me to believe that my ligation was not successful or that the
digestion did not work.
At this point I took a sample of Solenostemon scutellarioides from Wesley Wilson. The
following calculations were done using his gel in order to determine how much of the DNA
template needed to be sent as well as how much primer needed to be sent.
Primer amount=M1V1=M2V2
=(5pmol/ul)(50ul)=(200uM)V2
V2=1.25ul
Template= 3000bp/68000bp=4.41%
bp ruler=2ug * 0.0441=0.0882ug
4ul of DNA present in uncut band on gel
(0.044ug/4ul)=(0.011ug/1ul)
1.25ul of stock 200uM primer was sent along with 25ul of DNA template to the Virginia
Tech. laboratory to be sequenced.
I had a successful sequence in which I got data from the primers that annealed to the
plasmid(pJET SEQ R) and from the primers that annealed to the cloned GAPDH insert (GAP
SEQ F). For pGAPF, the first gene that came up, when I did an NCBI BLAST, was asparagus
officinalis. The pGAPF has a read length of 1531b, and once trimmed of 737b it was 794b. The
pJetR was also close to asparagus officinalis on NCBI BLAST. The read length was 1506b, and
once trimmed of 796b it was 710b. Both of these sequences are homology to the same GAPDH
gene. When I did a BLAST for the contig sequence, the first hit was Arabidopsis thaliana. The
contig sequence ended up being 1424b. The pGAPF was used to sequence in the forward
direction and the pJETR was used to sequence in the reverse direction. For the contig sequence,
we had a maximum depth of covered of eight sequences, which contained 377 bases.
Both of my individual sequences were successful. The following is the entire sequence
for my pJETR:
Any black areas are trimmed sequence and any red is gene sequence. There was a Q20 of 765b.
The following is the sequence of my GAPF:
Any are of blue is vector sequence and red is gene sequence. The Q20 was 736b for this
sequence.
The contig sequence was created from the entire class data. There was maximum depth
coverage of eight sequnces in some areas. There was a total of 1424b in the finished contig
sequence. Table 3.2 shows the mismatch updates that were made to get the final contig. There
were a total of eleven mismatched bases.
Table 3.2
Base
Before Change
After Change
73
-
G
75
C
G
516
A
A
605
-
-
606
G
G
680
G
G
682
A
A
706
A
A
707
A
A
909
T
T
1333
-
-
Table 3.3 shows how the sequences lined up as they were lined up and analyzed.
Table 3.3
Depth
4
8
4
3
Base to Base (Range)
1 to 539
8 to 916
917 to 1316
1362 to 1406
This table shows where all eight sequences lined up, which indicate which data is valuable and
which is more or less junk.
The top hit on NCBI BLAST for the contig sequence was Arabidopsis thaliana
chromosome 3. This indicates that our sequence closely matched the organism, Arabidoposis
thaliana, and it closely matched was chromosome three of that organism. The query coverage
was seventy-five percent, meaning that while the gene we sequenced was very similar to
Aradiboposis thaliana chromosome 3, it was not an exact match as we cloned a different gene
from a different organism. The following is the contig sequence that we compiled from all of the
class data:
V. Conclusion
The contig sequence we produced was between seventy and seventy-five percent similar
to the top five results done on NCBI BLAST. This indicates that, as a class, we were successful
at extracting, cloning, and sequencing the housekeeping gene for Solenostemon scutellarioides.
We successfully sequenced 1424 bases for Solenostemon scutellarioides.
The top five plants and genes which our sequence was the closest to was Arabidopsis
thaliana chromosome 3, Arabidopsis thaliana chromosome III, Arabidopsis thaliana cytosolic
glyceraldehyde-3-phosphate dehydrogenase, Asparagus officinalis cytosolic glyceraldehyde-3phosphate dehydrogenase, and Liquidambar styraciflua cytosolic glyceraldehyde-3-phosphate
dehydrogenase. The top three hits are from the same plant, Arabidopsis thaliana. It is widely
used model organism in plant biology and is a very common research organism. In fact, the
entire genome for this plant has been sequenced. There could also be a chance, since this is a
model organism, that our plant, Solenostemon scutellarioides, could have evolutionarily
evolved from Arabidoposis (2).
The samples I sent in to be sequenced were very successful as well. My pJETR had a
Q20 value of 765b, 796b trimmed, and 1506b total. My GAPF had a Q20 value of 736b, 737
bases were trimmed, and 1531 bases total.
I feel that as an individual, I was not successful as I was unsuccessful on multiple
occasions during this procedure. Mainly, I struggled getting PCR to work, which was likely not
my fault as several other classmates struggled with this as well. From this process I learned that
even the simplest biotechnology could be challenging and take several tries to complete
successfully. My time spent on this project has shown me that biotechnology is very interesting
and can be rewarding. I have learned a lot over this past semester from this class and this
project, including that biotechnology can be very beneficial if applied and monitored correctly.
VI. References
1)Barber, Robert D., Dan W. Harmer, Robert A. Coleman, and
Brian J. Clark. "American Physiological SocietyPhysiological Genomics." GAPDH as a
Housekeeping Gene: Analysis of GAPDH MRNA Expression in a Panel of 72 Human
Tissues. 8 Mar. 2005. Web. 24 Apr. 2012.
<http://physiolgenomics.physiology.org/content/21/3/389.full>.
2)"TAIR - About Arabidopsis." TAIR. Web. 25 Apr. 2012.
<http://www.arabidopsis.org/portals/education/aboutarabidopsis.jsp>.
3)"Solenostemon Scutellarioides." Missouri Botanical Garden.
Web. 25 Apr. 2012. <http://www.missouribotanicalgarden.org/gardens-gardening/yourgarden/plant-finder/plant-details/kc/a547/solenostemon-scutellarioides.aspx>.
4) Kit Summary. Biotechnology Explorer Cloning and Sequencing
Explorer Series Curriculum Manual. Bio-Rad. Catalog #166-5000EDU. Pages 1 and 2.
5)"FAQ About Genetic and Genomic Science." National Human
Genome Research Institute (NHGRI). Web. 25 Apr. 2012.
<http://www.genome.gov/19016904>.
6)"Cloning Fact Sheet." Oak Ridge National Laboratory. 11 May
2009. Web. 25 Apr. 2012.
<http://www.ornl.gov/sci/techresources/Human_Genome/elsi/cloning.shtml>.