Brindan Thiyagarajah

A61
Adam Cooke
Jared Gatto

Transformation, PCR, & Electrophoresis Lab

Introduction
These two sessions of lab demonstrated several key techniques and aspects of biology,
and DNA in general, such as PCR and Electrophoresis. The experiments involved the
transformation of E.Coli cells in different environments, essentially manipulation of the genes,
and then using such techniques such as PCR and electrophoresis to observe these changes. The
first half of the lab required the actual transformation of the E.Coli cells; this was done through
the application of certain independent variables: Ampicillin, and plasmids. Essentially, the point
of the ampicillin was to kill off the E.Coli cells, and leave behind ampicillin resistant mutants,
which then would be grown, the plasmids on the other hand were meant to house the E.Coli
from the ampicillin, and allowed cells without the resistant gene to survive in the ampicillin
environment. Ultimately, with these variables set in place, the growth of these E.Coli colonies
were observed, in four different scenarios: #1- no ampicillin and no plasmids (control group),
#2- yes ampicillin and no plasmids, #3- no ampicillin and yes plasmids, #4- yes ampicillin and
yes plasmids. The E.Coli was mixed in with a nutrient broth to promote growth, afterwards
placed in these different environments, and then were left to incubate until the next lab. The

second lab (Lab #10), is where the grown colonies of transformed cells would be observed in
order that the introduction of the resistant gene is confirmed. How is it observed exactly? Well,
since the gene sequence is known, two techniques known as PCR and Gel Electrophoresis can be
used. To start off, Polymerase Chain Reaction (PCR) is a method of analysis, where sequences of
DNA are copied through DNA synthesis and replication. In Lab 10, the E.Coli cells are going to
go through the same process of synthesis and replication; they will be broken in ssDNA and then
synthesized with primer and Taq polymerase, but since Helicase and Topoisomerase arent
available, the E.Coli DNA will need to be heated to the point where the DNA denatures into
single strands, and then is synthesized by the primer and Taq polymerase from there on. In the
experiment, a PCR premix, was distributed, which contained the Taq polymerase, essential salts
and buffers, and dNTPS (dATP, dCTP, dGTP, and dTTP). The Taq polymerase and dNTPs,
work along with the primer (phage m13 oligonucleotides) which is then later added, to assist Taq
polymerase. As its known, DNA polymerase works as an extender, it cannot start off a
sequence, so we have the primer in the solution to set a base template (5
GTAAAACGACGGCCAGT 3 and 5 CAGGAAACAGCTATGAC 3) where the polymerase
can work off of. The second half of the lab was the use of electrophoresis to identify the number
of DNA that has migrated from a well, located on the gel. By using UV rays, several DNA
bands can be seen, which show how far the DNA has migrated. Essentially the further strand
the more DNA there is. But how is one supposed to tell the number just by looking? Molecular
weight standards that contains several DNA of known value and high conductivity are used in
comparison with the DNA that is being measured; the molecular weight standard will be ahead
of the E.Coli DNA due to its conductively, and once the procedure is done the E.Coli DNA will
be compared to that of the Molecular weight standard so that an estimate number can be

produced. Knowing the number, will allow us to know whether or not the DNA contains the
resistant gene. By using these techniques scientists are able to discover new things about
biological life every day. Just humans alone have 24,000 genes, and electrophoresis and PCR
enables humans to find out what a particular gene is capable of, whether its detrimental,
provides resistance, etc. It is because of electrophoresis, and PCR, a scientist is able to identify
and isolate specific genes and find out its purpose, even potentially creating a vaccine, for
example is a possible outcome.

Hypothesis
Part A:
It is hypothesized that plate one containing neither ampicillin nor plasmids will lead to an
immense overgrowth of cells to the point there aren't any colonies but a densely collected and
large abundance of cells; reason being because there is no ampicillin being introduced to the
environment, allowing the E.Coli cells to replicate freely without dying to the ampicillin. As for
plate two, there will potentially be no cells at all because the ampicillin that is present will kill
them all, except for the very few cells that have an ampicillin resistant gene. Plate three, will be
very similar to plate one simply because there is no ampicillin present so the cells can replicate
freely in the nutrient broth, but they will not utilize the plasmids because that requires an
immense amount of energy, whereas E.Coli cells likes to be energy efficient; it is quite possible
that plate three will contain a dense collection of E. Coli cells along with plasmids. As for plate
four, it is predicted that it will have some growth of E.Coli, depending on the colonies found on
the plate; for example, if the plate contains ampicillin resistant E.Coli they will replicate without

the use of a plasmid simply because it consumes too much energy, and as a result the plate will
have colonies of ampicillin resistant E.Coli cells, but you will also have colonies where E.Coli
cells without the resistance gene, and in turn will have to use the plasmids in order to survive in
the ampicillin. The colonies for the E.Coli cells which have to use the plasmids will probably be
less in number simply because they will consume more energy to replicate compared to a cell
with the resistance gene. And one also has to take into account that not all the E.Coli cells will
transform along with the plasmids.
Part B:
It is hypothesized that in the PCR process and Gel electrophoresis that +amp and amp
samples of E.Coli will naturally have a ratio of lower concentration of cell, to higher
concentration of cells. Reason being for this hypothesis is due to the fact that, in the +amp tube
there will be E.Coli cells which were once exposed to ampicillin, and the cells that are in tube are
either are or descendants of the surviving E.Coli cells that didnt die off to the ampicillin; hence
it will consist of E.Coli that utilized plasmids, and/or E.Coli that has the ampicillin resistant
gene. It is also predicted that a majority of the cells in the +amp tube utilized the plasmids to
survive in the ampicillin, and as a result they will consume more energy which in turn will slow
down the replication process. However, the amp quite likely will have a higher cell count
simply because the cells will not be killed off and will be able to replicate at a normal pace, and
as for the plasmids they simply wont be used by the cells because they are simply inept for
energy consumption. With these predictions at hand, it can also be assumed that in
electrophoresis the +amp will form a band that exhibits a color under an ultra violet light, simply
because the cell expresses the gfp gene, and then this will be visible under ultra violet. The fact
that in the +amp solution the cell is transformed further proves that a color will be emitted,

because the ampicillin will force many cells to transform in order that they may survive, and as a
result these transformed cells will express GFP which is apparent under UV lighting. Knowing
this, it can also be assumed that in the amp solution, there will be no GFP being expressed
because there is no need for the cells to transform in the solution, therefore GFP will not be
produced nor expressed.
Methods & Materials
Lab 9
Materials: 50 mM CaCl2 Microfuge tubes, Assorted Micro-pipettes, E.Coli culture nutrient agar
plates, inoculation loops, ice, water bath (42oC), 250 L Nutrient broth, four nutrient agar plates
In Lab 9, start of by taking two microfuge tubes containing 50mM CaCl2, and label one
pGreen and the other +pGreen. Next step is to pipet 10 L of pGreen plasmid into the +pGreen
tube, once thats completed, an inoculation loop is required to transfer E.Coli cultures from agar
plates into the microfuge tubes. Mix the content of the tube, and then ice it for 15 minutes, while
placing the inoculation loop back into its sterile sleeve. Once the 15 minutes have elapsed, you
need to heat shock the cells in order that the cells pores open up and allow the plasmids in, and
to do so place the tubes into 42oC water bath for 50 seconds precisely, and afterwards the tubes
need to be dropped into an ice-water bath for 2 minutes; this temperature change will cause a
rapid shrinking that acts like a vacuum for the plasmids to be absorbed into the cell. Once
completed, take 250 L of nutrient broth, and add it into both microfuge tubes by using a p200
micropipette (125 L at a time), and then let the solution sit for 10 minutes in room temperature.
While the solution is idly sitting at room temperature, four nutrient agar plates should be
obtained, each should be labeled respectively: #1-amp/-p, #2+amp/-p, #3amp/+p, and

#4+amp/+p. Using the inoculation loop and micropipette, pipette 100 L of pGreen tube into
plate #1, then spread it with the loop: repeat the step for plate #2. As for plates #3 and #4, deposit
100 L of the +pGreen tube content into each plate respectively, and use a new inoculation loop
end to spread it throughout each plate. Take the plates, bundle them together, and put them in the
fridge in order that the cultures on the plates may incubate.
Lab 10
Materials: Ultra violet light, two 1.5 mL microfuge tubes, distilled water, inoculation loop, heat
block (95oC), ice, two .5 mL microfuge tubes, PCR Premix, Primer solution, PCR thermal
cycler, Agarose, gel casting tray, gel box, leads, power supply, TBE buffer, +amp and amp
tubes, tracking dye, molecular weight standard, gel documentation system
The E.Coli cultures acquired in Lab 9, will now be observed, and so they must be taken
out of the refrigerator, and then placed underneath ultra violet light; whichever plate has colonies
emitting a fluorescent green color should be recorded.
PCR: For the PCR procedure, two 1.5 mL microfuge tubes should be obtained and then filled
with 50 L of distilled water, then respectively labeled +amp and amp. Taking a sterile
inoculation loop, open the plate with the fluorescent green colonies (+amp/+p #4 plate) and
acquire a colony on the inoculation loop, then transfer it to the +amp microfuge tube. Next take
plate #3, and repeat the steps prior, but this time place it in the amp tube. Take these microfuge
tubes, and place them in a 95oC heat block for 5 minutes so the DNA can denature and disperse,
then cool the tubes in an ice-water bath. Next step is to obtain two more microfuge tubes, but that
are .5 mL each and containing 40 L of PCR Premix, and label these respectively +amp and
amp. Furthermore, take the PCR Premix tubes, and add 5 L of the solutions that were

previously heated, into their appropriate tubes. In addition, to this solution, add 5 L of primer
solution to both tubes. Finally, place the microfuge tubes into the PCR thermal cycler for 3
hours.
Gel Electrophoresis: For the gel electrophoresis, agarose gel will first need to be acquired from
the 60oC water bath. Then at the lab bench, a gel casting tray will be available, and the flood
gates of the casting tray must be secured in order to proceed to the next step. Place a comb at the
far end of the tray, and then place the tray on a leveled platform where it wont be interfered
with. Pour the agarose onto the tray, and let it solidify which will take around 20 minutes. Next,
loosen the gates and take the tray and place it in the gel box so that the wells formed by the comb
are facing towards the cathode. Take 300 mL of TBE Buffer and pour it into the gel box, while
removing the comb from the gel. Take the +amp and amp tubes, and add 10 L of tracking dye
to each. Then take 25 L of Molecular weight standard, pipette it into one of the wells, do the
same for the +amp and amp (each sample should have their own respective well). Put the cover
on the gel box, connect the leads to the power supply, and set the voltage to 100V - 120V. Run
the experiment until the tracking dye has gone or more of the gel, which is approximately 4560 minutes. Once done, turn off the power supply, take out the leads, and take the gel out, and
put it on a weight tray. Take the gel, and place it under ultraviolet light and then DNA bands
should become apparent, and using the gel documentation system, take a print out of the gel.

Results
Figure 1: Table 1: Lab 9 Plate Variables

Plate Number

Plate Content

Fluorescent

Number of Colonies

-amp/-p

No

TNTC

+amp/-p

No

-amp/+p

No

TNTC

+amp/+p

Yes

24

In figure 1, the table shows the relationship of ampicillin presence and plasmid presence,
to the number of colonies formed, and fluorescence. Plate 1 had no ampicillin and no plasmids,
so the cells were able to grow freely without persecution, and so they ended up amassing to a
large amount, so large that it wasnt countable, but it wasnt fluorescent because the cell did not
transform with plasmids which provide the fluorescent color. As for Plate 2, there was presence
of ampicillin, but there were no plasmids introduced to the solution, and so the E.Coli cells were
killed off by the ampicillin, hence no fluorescence and no colonies either. Plate 3, like Plate 1,
had no ampicillin, but Plate 3 did have plasmids which generally had no purpose for the cells,
mainly because they werent exposed to ampicillin so the cells werent at risk therefore they
didnt need the plasmids to survive; ultimately, the cultures on Plate 3 replicated so much that it
was too numerous to count like Plate 1, and since the plasmids werent utilized by the cells, GFP
couldnt be expressed, therefore no fluorescence. Finally, Plate 4, was exposed to ampicillin, and
plasmids, meaning that cells that accepted the plasmids were transformed and had the ability to
survive in the ampicillin, and at the same time these cells will also express GFP, meaning they
had fluorescent. The reason why Plate 4, had a countable amount of colonies is because not all
the cells transformed.

Figure 2: Gel Electrophoresis reading

In figure 2, each lane can be observed to contain molecular weight standard, +amp, and
amp respectively. Using the molecular weight standard, the number of base pairs and molecular
weight of the samples can be determined. The bands on the gel also provide evidence that a gene
is present, for example +amp has bands visible under the UV lights meaning the gene is present,
whereas amp isnt apparent, or is barely distinguishable meaning the gene is not present. By
comparing the +amp with the MWS, it is estimated that the gfp gene has approximately 850 to
900 base pairs.

Figure 3.

Migration Coefficient vs. LogBP

0.9

Migration Coefficient

0.8
0.7

y = -0.3714x + 1.706
R = 0.9727

0.6
0.5
0.4
0.3
0.2
0.1
0
0

0.5

1.5

2.5

3.5

4.5

LogBP

In figure 3, the Log of the Base Pairs is being compared to the migration coefficient. The
migration coefficient was found by dividing distance of the dye front from the well (8.5cm) by
the MWS bands: each one of those values gave the migration coefficient, and as for the Base
pairs, the log was just simply taken of each base pair band on the MWS. Compiling the data
together, results in figure 3, and with the trend-line equation given in figure 3, x can be found:
x representing the number of base pairs found in the +amp DNA sample. By finding the
distance of the +amp band from the well (5.2 cm), and then dividing that by the dye front
distance from well (8.5 cm) you get the migration coefficient for the band on +amp. This
migration coefficient can then be plugged into the trend line formula as the y value, and then x
can be solved for. In this case, x = 2.9462 which is also the LogBP, and so to get the number of
base pairs simply do, 10X which in this case is 102.9462= 883.49 base pairs. The fact that the

data on figure 3 had also had an R2= .97, further strengthens how accurate the results are, simply
because the closer the R2 is to 1, the more accurate your results.

Discussion
The data values that were recorded and experimentally found, do happen to correlate as
well as agree with the hypotheses given prior: since the gfp gene is apparent in the +amp sample
electrophoresis bands, the hypothesis that the +amp would express the GFP because the cells had
transformed along with the plasmids holds veracity. Clearly, if this werent the case, there
wouldnt be any DNA bands showing up under the UV light. The growth on the plates, had also
further proved the hypotheses correct; it was stated earlier that the plates each had differing
results because of the combination of variables, so for example plate one having no ampicillin
and no plastids reciprocated by having an abundance of E.Coli cultures growing on the plate,
whereas plate two had ampicillin but no plastids, and the result was that there would be no
cultures because the ampicillin would have killed them off, which happened to be the case. As
for the PCR product, the base pairs were relatively close to 750, but slightly off: the reasoning
behind the discrepancy can quite possible be the cause of human error, for example, if too many
plasmids were added to the +amp solution, then it is possible that more than intended cells
transformed, meaning more GFP, and then a higher base pair count.
Conclusion
In conclusion, it was seen that gfp gene was found to be in the +amp sample, compared to
the amp which didnt have the gfp gene, and the fact that these bands can be seen under UV
lights further goes to show that gfp gene is actually apparent in the +amp sample. Since it is

known that the +amp sample contains the gfp gene, it is also correct to assume that the
hypotheses given earlier about +amp and amp were correct: in laymans terms, it was
hypothesized that the +amp would have the gene whereas amp wouldnt, simply because on
culture decided to use the plasmids where the other didnt.

Work Cited
-

Lawless, Jeanne BIOL 118 Lab Manual