South’s Waksman Club
Kiran Vaidya
Waksman Project Overview
What is the purpose of Waksman?
To learn the latest molecular biology techniques and to sequence and analyze the cDNA clones
from Wolffia australiana (Duckweed) as well as compare the sequences to other species.
*The latter part basically means we want to obtain only transcribed DNA that most likely
serves a function in Duckweed then sequence and analyze it. One way we analyze this
DNA is by comparing it to the DNA of other species.
The sequences of the most functional portions of the genome may be very useful to scientists who
are studying Duckweed which may have some potential uses for society.
These are the reasons the Waksman Student Scholars Program chose Duckweed.
 Potential biofuel source
o Under cold temperatures can accumulate 40%-70% starch
o Sinks to bottom of ponds
o Starch can easily be converted to sugar for fermentation
o Will not compete with food crop production
 Bioremediation
o Grows in contaminated water
o Sequesters or degrades contaminates such as lead, arsenate, halogenated
compounds
o Extracts nitrogen and phosphate from waste water
 Potential food source
Our research project involves sequencing genes from Duckweed and analyzing it. In our
analyses, we determine the functions of these genes. If the analysis is correct, it is then sent to
the NCBI to be published on DNA sequence databases.
What is our job as members of the club?
To identify insert sizes of cDNA clones by PCR, RD, and gel electrophoresis. After sequencing the
clones, we analyze the DNA using bioinformatics.
*So, we basically need to determine the size of the DNA, and purify the DNA. Then after
the DNA is sequenced, we analyze it on DSAP.
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Kiran Vaidya
Overview of the Project:
Step 1: Construct a cDNA library
*This is done by the WSSP staff
Can we sequence all the DNA from Duckweed?
 No, we have to be selective. Not all DNA serves a function so we only want to
sequence the DNA that will most likely serve an important function in Duckweed.
Problems with sequencing the entire genome:

Because Wolffia australiana (Duckweed) is eukaryotic, its mRNA transcripts go
through mRNA splicing. So if we sequence DNA from Duckweed directly, its
sequence won’t be functional because its mRNA transcripts contain introns.

The genome itself has promoters, intergenic regions, and non-expressed genes.

Only a small percent of DNA codes for protein.
So how can we get DNA that is functional?
We can’t just extract DNA because then we won’t know which sections do or do not serve a
function. So instead, we extract mRNA transcripts that have already been spliced and we use
reverse transcriptase (RT) to get functional DNA that code for proteins and that doesn’t have
introns.

This DNA is called cDNA.
Now that we have cDNA, why can’t we sequence it?
Problems:
 Only one copy of the sequence, not enough DNA for sequencing, we need to get
more copies.
 We need to determine the size of the insert first. If the DNA has an insignificant
number of base pairs, it most likely won’t code for a full functional protein. We
might have gotten a partial mRNA transcript when making the cDNA.
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Kiran Vaidya
In order to study a DNA fragment, it needs to be amplified and eventually purified.
 We can do this by cloning the DNA into plasmid vectors which are put into
bacteria. These plasmids can replicate in bacteria such as E. coli.
 These bacteria then reproduce to form colonies. Each daughter bacterium cell in
a single colony is identical to the parent and contains the same plasmid with the
same insert. This is why they are commonly referred to as clones.

Each bacteria colony has a different insert.
Step 2: Growing the bacteria cultures (Overnights)
We pick bacteria that have been transformed (contain inserts) and grow them overnight
to obtain a larger number of cells than the original colony. This amplifies the DNA for the
minipreps.
Step 3: Polymerase Chain Reaction
Purpose: To amplify the insert DNA even further to get a good band in a gel.
We allow replication to occur in a test tube by supplying the tubes with materials for
replication and cycling them through different temperatures to allow for PCR to occur.
PCR makes it easier to determine the size of the insert from the gel in the next step.
Step 4: Gel electrophoresis
Purpose: To determine the size of the insert.
If we get nice bright bands, we know that the PCR worked and we amplified the DNA.
Then we determine the size of the insert by comparing it to a DNA marker containing
DNA of known size in the first lane.
Step 5: Minipreps
Purpose: To purify the plasmid DNA for sequencing of the insert.
We do this by harvesting the cells, lysing the cells, and removing all impurities such as
the bacterial chromosome, lipids, proteins, etc.
Step 5: Restriction Digest and Gel electrophoresis
Purpose: To verify that the right plasmid has been purified.
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Kiran Vaidya
By cutting the plasmids from the minipreps with restriction enzymes, we can run a gel
with the resulting DNA fragments. Then we can add up the sizes of the DNA in each
band and subtract the size of the plasmid vector and verify the size of the insert. We only
use some DNA from the miniprep to do this step. If we know the DNA is good enough,
we send it in the minipreps for sequencing.
Step 6: Send the DNA for sequencing and analyze the sequence
We send the minipreps to get sequenced and we then analyze the sequences on DSAP.
DNA Review:
Function: DNA itself does nothing other than hold all the information the body needs to function.
This information goes towards making proteins. Proteins carry out all the major functions of the body.
DNA helps the body function by telling it how to make each specific protein. It does not make the
proteins itself. It just contains the information that the body uses to function.
Structure of DNA:
 DNA is a double stranded molecule.

Its strands are anti-parallel

It has a 5’ end and a 3’ end

Both Strands are complementary
What does a 5’ end and a 3’ end mean?
DNA is a polymer made of nucleotide monomers. These
monomers consist of a deoxyribose sugar, a phosphate group
and a nitrogenous base. The carbons in the sugar are numbered
from 1-5. In a nucleotide, the 5’ carbon binds to the phosphate
group. The 3’ end is left free with an OH group bound to it.
When nucleotides come together, the 3’ carbon binds to the
phosphate group on another nucleotide to form a polymer. So
after a polymer is formed, one side will have the 5’ carbon on its
end and the other side will have the 3’ carbon on its end. The
two strands will have different numbered carbons on their ends
when they match up as seen in the picture. This is why the DNA
strands are said to run from 5’ to 3’ and the two strands are antiparallel to one another. Nucleotides can come with 4 different
bases: Adenine, Thymine, Guanine and Cytosine. They can
combine in any order and together they make the unique DNA
sequence of each organism.
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Plasmids vs. Linear DNA:
DNA can come in two forms: linear or circular. Eukaryotes have linear DNA. Typically,
prokaryotes have circular pieces of DNA called plasmids in addition to their main circular
chromosome.
Plasmids can replicate inside of bacteria and can be manipulated to do what we desire. The
plasmid vector we use is of known size. We also know the genes that are located on it and several
sequences of DNA that can be very useful to our experiment. This includes the lacZ gene, the
Ampicillin resistance gene, and the two PvuII sites. Their importance will be explained later as
each lab is explained. It is important to realize that the plasmid comes to us already with the
insert and is already inside the bacteria. This is done by the WSSP staff. But we don’t know for
sure whether the plasmid was successfully inserted into the bacteria or whether the insert was
successfully inserted into the plasmid vector. We need to select bacteria that were successfully
transformed with the plasmid vector
and insert cDNA before continuing.
Replication: Replication is the
process by which DNA makes more
copies of itself. It is semi-conservative
meaning that one parent strand is
conserved along with a new strand in
each new copy of DNA. Replication
starts at the origin of replication. In
plasmids, there is only one origin of
replication.
Steps:
1.
DNA unwinds (by helicase)
2.
Replication Fork (bubble) Forms
3.
DNA polymerase brings in nucleotides in the form of
nucleoside triphosphates to conduct dehydration synthesis
with the existing nucleotide. (*problem: DNA polymerase
cannot start replication. It can only add to existing nucleotides.
This is why we need primers. Primers are small sequence of
RNA nucleotides that bind to the first few bases of the DNA.
DNA polymerase continues replication by adding to
nucleotides to the existing primers. The primers are cut out
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later after replication is completed and replaced with the proper DNA nucleotides.)
4.
Since DNA polymerase can only work in the 5’ to the 3’ direction, one strand will be
the “leading” strand that undergoes continuous replication. The other strand will be
“lagging” and replicated in fragments. This strand needs multiple primers.
5.
After the two daughter strands are made, they rewind.
*Note that the primers on the 5’ ends of the strands cannot be replaced as DNA polymerase
cannot work in the 3’ to 5’ direction to add nucleotides and fill in the empty spots. Therefore,
DNA will get shorter and shorter every time it replicates but telomeres with noncoding DNA
help avoid deleting important regions.
Transcription:
DNA
RNA
1.
DNA unwinds
2.
RNA polymerase creates a complementary strand
made of RNA nucleotides to hold the information
held by the coding strand of DNA. It uses the
Template strand (the 3’ to 5’ strand) of DNA in order
to do so. This RNA strand is called the pre-mRNA
transcript.
3.
After the information of DNA has been transcribed
or “copied” onto an RNA molecule, the DNA
molecule rewinds. At this point, the pre-mRNA
transcript is not functional.
mRNA Splicing:
The mRNA transcripts contain introns which are regions that are
not translated into proteins.
These regions need to be spliced out of the transcript.
Splicesomes (made of RNA and protein) cut out these introns.
Then the exons (regions that are translated into protein) come
together and create the functional mRNA transcript. Added onto
the transcript is a methylated guanine cap at the 5’ end and a poly A
tail at the 3’ end. (The main purpose of the tail is that the mRNA
transcripts are degraded once when they enter the cytoplasm. The
tail gives the sequence some time to be translated before it is
degraded.)
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Kiran Vaidya
The mRNA transcript then travels
out of the nucleus and to the
cytoplasm.
Translation:
 The small ribosome subunit
attaches to the mRNA transcript and
locates the start codon (the sequence
AUG).
 The appropriate tRNA
molecule with the matching
anticodon (complement) of the start
codon binds to the transcript.
 The large ribosomal subunit
attaches as well and translation
begins.
 The mRNA molecule is read
in groups of 3 bases called codons.
The open reading frame is
established by the start codon. Each
codon has an anticodon. tRNA
molecules with the appropriate
anticodon bind to the mRNA and
bring amino acids along. As each
tRNA molecule begins to leave, it
breaks its bond with the amino acid. This energy goes into forming the peptide bond between
adjacent amino acids. This process continues until the polypeptide is formed and the ribosome
reaches the stop codon (which has no matching tRNA molecule). A release factor binds instead
causing the whole complex to dissemble. The polypeptide that is formed will be folded to
become a functional protein.
Restriction Enzymes: Restriction enzymes are used by bacteria to fight viral infections. Viruses work by
injecting their DNA into a host cell and incorporating the DNA into the host genome. Viral proteins are
then transcribed and lead to the cell lysis (breaking down of a cell) and the production of more viruses.
Restriction enzymes recognize certain DNA sequences and cut them at
specific locations to render them useless. Restriction enzymes will cut
the particular sequence they recognize no matter where they see it. So,
bacteria protect their own genomes by methylating their DNA.
Restriction Enzymes are essentially molecular scissors that we can
use to cut DNA at specific sites.
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Kiran Vaidya
Waksman labs:
Growing the bacteria cultures (Overnights):
We need to know if the bacteria were transformed and contain the insert.
So we need to grow them in culture and test for the presence of the insert.
Screening For Inserts:
 The bacteria themselves aren’t ampicillin resistant. In this case, if we grow
them in media containing ampicillin, these bacteria would die.
 However, our plasmid vector has the ampicillin resistant gene. So any
bacterial cell that took up the plasmid will transcribe and translate this gene
and become ampicillin resistant. Only these cells can grow on the petri dishes.
o
Conclusion: We know that any cell that grows into colonies on
the petri dish has the plasmid vector.
 The plasmid vector also has the lacZ gene which codes for Beta-galactosidase.
This protein is responsible for breaking down lactose when glucose levels are
low.
 However, Notice that the site where the plasmid is cut and where the insert is
inserted is right in the middle of the lacZ gene.
 So, plasmids that have the insert, cannot transcribe the lacZ gene because the
gene is interrupted by the insert whereas plasmids without the insert can
transcribe the gene.
 We use this as a marker by putting X-gal (a molecule similar to lactose) in the
petri dishes. Bacteria with plasmids containing inserts cannot break down Xgal because they cannot transcribe beta-galactosidase to do so. These bacteria
remain white. Bacteria with plasmids without inserts can transcribe betagalactosidase and break down X-gal. The result is that these colonies turn blue.
o
Conclusion: We know that blue bacterial colonies contain
plasmids without inserts and that white bacterial colonies
contain plasmids with inserts. We should only pick bacteria
from white colonies.
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Grow Colonies:
Then we grow the colonies overnight in LB broth containing ampicillin
(to make sure only the bacteria containing the plasmid with the insert grows).
This way, we obtain a larger number of cells. In obtaining a larger number of
cells, we are effectively amplifying the amount of plasmid DNA that we have.
We put the tubes containing the bacteria and broth into a shaker at school which
aerates the broth and allows the bacteria to grow.
We can now go two ways! As shown below.
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Kiran Vaidya
Polymerase Chain Reaction:
*it makes the most sense to do PCR, run a gel and determine the size of the insert
to see if the DNA sequence is long enough for sequencing. If not, there is no point in
wasting materials and purifying the DNA.
In order for this reaction to occur, there are several important materials we need.
We need: nucleoside triphosphates, primers, the DNA, and DNA polymerase.
We avoid the need for helicase and other enzymes in this procedure. We try to keep it as
simple as possible.
1. We make a primer mix for 5 people. This mix contains double distilled water, and the
forward and reverse primers that are required to initiate replication on both DNA strands of
the insert on the plasmid. We don’t want to replicate the whole plasmid, just the insert. By
only providing primers for the insert, only the insert will be amplified and the plasmid
vector will be of insignificant amount. (Note that these primers are made of DNA not RNA so
there is no need to cut them out later).
2. Then we obtain tubes with a PCR bead which has taq buffer, taq polymerase and the
nucleoside triphosphates. (Note that we don’t use normal DNA polymerase because in order
to allow replication to occur, we subject the DNA to high heat. This heat would denature
DNA polymerase so instead we use taq polymerase found in thermophilic bacteria. High
heat does not affect protein activity in these thermophiles as they are naturally found in hot
environments.)
3. We put all these materials in the same tube and put it in the thermal cycler.
Thermal cycler:
1.
Initial Denaturation
We set it at 94oC for 5 minutes (This lyses the cells and denatures the DNA into two
separate strands).
2. Amplification (The thermal cycler will repeat these three steps in order 30 times.)
94oC for 30 seconds (Denatures the target DNA-insert )
52oC for 30 seconds (The primers then attach to the insert)
72oC for 1 minute (nucleotides are added to form a new DNA strand)
*By repeating these steps multiple times, we allow for replication to occur
continuously. Once a new daughter DNA molecule is formed, it is immediately
denatured so that it can replicate once more.
3. Additional Elongation (This step insures all DNA strands are full length)
72oC for 5 minutes
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IV End Program After step 3, the thermal cycler will drop the temperature of the heating block to
4oC so that they are in a stable environment.
PCR Gel electrophoresis: Determining the Size of the insert
*Recall that DNA is composed of a sugar, a phosphate group and nitrogen base. The phosphate
group has a negative charge and therefore makes DNA itself negatively charged. Since negative
charges and positive charges attract, DNA will migrate toward anything that is positively
charged.
Now that we have performed the PCR, we have enough DNA to determine the size of the DNA
fragments. In doing this, we want to separate the DNA by size. By putting a solution of DNA
that is negatively charged into an electric field, the DNA molecules will migrate toward the
positively charged electrode. This migration pathway is provided by the tiny pores of the
agarose gel that we put the DNA into. Larger fragments have more trouble going longer
distances through the gel and therefore do not migrate very far. Shorter fragments migrate the
farthest from the origin as they don’t have as much trouble moving through the tiny openings.
We put this gel into a gel chamber with an electrical field. We load the DNA into the gel into
wells at the negative end of the gel. After a short time, the DNA fragments will have separated
by size. DNA of similar size will have moved the same distance and will be visible as bands. We
add loading dye to the solution to ensure the DNA has sunk to the bottom of the well (instead
of floating away in the buffer) and to see if the gel is working by seeing the migration of the
loading dye. However, this dye does not stain DNA. Ethidium Bromide (already in the gel)
stains DNA and allows it to fluoresce when exposed to UV light.
After the DNA has migrated, we put the gel in a UV box and take a picture of it. We can then
determine the size of the DNA by comparing it to a DNA marker with fragments of known
sizes which we load in the first lane. By comparing the distance migrated by DNA in the marker
and the distance migrated by our DNA, we can determine the size of the insert.
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Minipreps: Purifying the plasmid DNA
*After determining if the insert is large enough to be sequenced, we purify the DNA,
verify the size of the insert, and send the DNA in for sequencing.
1. Harvest the cells by centrifuging the microcentrifuge tubes containing the
bacteria culture.
a. The cells will collect in the pellet as they are the densest substance. We discard the
supernatant because it just contains LB broth.
2. Add solution I –resuspension buffer, RNase.
a. We are resuspending the cells, and degrading any RNA fragments (which would
make it difficult to purify the DNA).
3. Add solution II- lysis buffer, NaOH.
a. Here we are lysing the cells and putting them in a basic solution. This basic solution
denatures all double-stranded nucleic acids making them single stranded (plasmids
and chromosome).
4. Add solution III-potassium acetate then
centrifuge.
a. Potassium acetate is acidic and therefore allows the
DNA strands to come back together. Because the
chromosome is very large, it cannot fully renature
whereas only small DNA strands such as plasmids
can renature easily. Plus, salts will start forming
which can act like a net and pull down the
chromosome to the bottom of the tube when we
centrifuge.
5. Save the supernatant which contains the plasmids
and discard the pellet which contains proteins,
the cell wall, the chromosome, etc. Pour the
supernatant into a spin column.
6. Centrifuge again and pour out the flow through which is additional
impurities that is not need.
7. Add Wash Buffer –ethanol and centrifuge again
a. Which gets rid of any remaining impurities
b. Then centrifuging it again gets rid of the ethanol
c. Discard the collection tube and replace with a microcentrifuge tube
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8. Add elution buffer and centrifuge
a. Which elutes (removes) the plasmids from the spin column into
microcentrifuge tube.
You now have purified plasmid DNA.
Restriction Digest:
*Recall that restriction enzymes can be used as molecular scissors to cut DNA at specific sites.
Most restriction enzymes recognize symmetric inverted repeats of base pairs called
palindromes.
When we set up for a restriction digest, we need to have the right restriction digest for our
plasmid, buffer, ddH2O, and loading dye.

We will use the enzyme PvuII. Our plasmid vector has two PvuII restriction sites where
this enzyme can cut the plasmid.
 By cutting it the DNA into fragments, we have created another way of verifying the size
of our insert.
 The plasmid vector is of known size. So by allowing the DNA to be digested by the
enzyme and then running the DNA in a gel, we can size out each of the fragments.
 Then we can determine the size of each fragment by comparing it to the DNA ladder.
 Then we can add up the size of each fragment and subtract the size of the plasmid vector
to get the size of our insert.
*note sometimes digestion is incomplete and some plasmids will not get digested. This can lead to
additional bands in the gel formed from plasmids migrating by themselves or plasmids getting tangled
up forming supercoils.
Why do you do a restriction digest of bacteria that you have already done
PCR on and verified that the insert is present?
 You do this because you want to make sure that you did purify the plasmid correctly.
Sequencing is an expensive process and you don’t want to sequence something that did
not work.
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After verifying that the insert is long enough to get sequenced, we
send our Minipreps in for sequencing. Then we analyze the
sequences on DSAP.