electrophoresis and restriction digestion, 3 questions.

electrophoresis and restriction digestion, 3 questions.

electrophoresis and restriction digestion, 3 questions.

Appendix C: Use of quantitative DNA ladder 2014
Restriction Digestion and Gel Electrophoresis
R6. Show a figure with the picture of your gel (step 33) and the corresponding labels.
Estimate the migration distance of all bands. Plot logMW (bp) of the MassRuler DNA
ladder (figure 3 and Appendix C) vs. the migration distance (show the figure). Estimate
the MW (bp) of all DNA fragments in your four samples.
(BELOW IS THE GEL, further below is background information and and
procedures, plus appendix)
label first well ?Eco, Hind, E+H, Control, Ladder

Ladder

R7. From the number and size of the fragments observed in your three digested
samples, create a map of the plasmid showing the relative location of the restriction
sites for EcoRI and HindIII.
R8. Looking at your gel electrophoresis identify the format(s) your the undigested
plasmid has adopted? Were your digestions (EcoRI and HindIII) complete? Why or
why not?

F. Plasmid

Appendix C: Use of quantitative DNA ladder 2014
Plasmids are circular, double-stranded DNA molecules that exist independently of
chromosomal DNA in bacterial cells and can range in size from 1-200 kilobases. These
plasmids can be stably inherited by the daughter cells following replication or
transferred to other bacteria by a process known as horizontal gene transfer (HGT). A
common form of HGT is called “conjugation”, where bacteria can transfer plasmids by
direct cell-to-cell contact. Most naturally occurring plasmids carry genes that provide an
advantage to its host, such as antibiotic resistance or the production of specific
enzymes.

Scientists have taken advantage of plasmids in order to manipulate genes. These
experimental plasmids, also referred to as vectors, have undergone a variety of
modifications in order to make them more useful to scientists. They can be used to
clone and transfer genes, as well as for the expression of recombinant proteins. In the
laboratory, plasmid transfer is achieved by temporarily rendering cells permeable to
small DNA molecules, using chemicals (such as calcium chloride, a source of divalent
cations) or electrical shock. Plasmids under “relaxed” replicative control may be present
in anywhere from 10 to over 500 copies/cell, thus allowing the host to produce a large
amount of plasmid DNA from a given number of bacterial cells, which is especially
useful for cloning purposes.

The types of plasmids used in the lab usually contain at least the following features
(figure 2):
i) Origin of replication: Allows the plasmid to replicate independently of the
bacterial cell’s chromosomal DNA . This is the DNA sequence that will be
recognized and bound by the replication machinery.

ii) Multiple cloning site (MCS): A short stretch of DNA that contains recognition
sequences for several different restriction enzymes. This allows us to insert
fragments DNA, such as our genes of interest, into the plasmids. It is important
to note that the restriction enzyme recognition sequences found within the MCS
are unique and are not found elsewhere on that same plasmid. When the
plasmid is replicated, so is the inserted DNA, allowing for amplification of this
newly introduced sequence.

Appendix C: Use of quantitative DNA ladder 2014
iii) Selectable marker: It is impossible to visually tell the difference between a
bacterial cell that carries a plasmid and one that does not. That is why plasmids
used in the lab have been engineered to contain a selectable marker, a gene that
imparts a certain trait to the bacteria and allows us to tell whether the cells
contain our plasmid of interest. A gene that confers antibiotic resistance
(ampicillin-resistance is commonly used) is often found on these types of
plasmids. Only the bacteria that contain the plasmid will express the antibioticresistance gene, allowing them to survive and replicate in an environment
containing ampicillin. Bacteria that do not contain the plasmid will not survive.

Figure 2. Plasmid DNA. Schematic
of a generic plasmid depicting
their common features: an origin of replication, the multiple cloning site (MCS) with a
density of unique recognition sites for restriction endonucleases and a gene conferring
antibiotic resistance, the most commonly used selectable marker.
G. Agarose Gel Electrophoresis of DNA
Gel electrophoresis is a technique used to separate macromolecules - especially
proteins and nucleic acids - that differ in size, charge or conformation (6, 7). When
charged molecules are placed in an electric field, they migrate toward either the positive
(anode) or negative (cathode) electrode according to their charge. In contrast to
proteins, which can have either a net positive or net negative charge, nucleic acids have
at neutral pH a negative charge, due to the phosphate groups of their backbone. The
relative migration distance of each molecule is determined by the charge density of the
molecule and the resistance of the matrix (or gel) media to the passage of the molecule.
In DNA/RNA agarose electrophoresis (8), a gel of agarose is cast in the shape of a
horizontal thin slab, with wells for loading the sample close to the cathode (usually
connected to the black wire). The gel is immersed within an electrophoresis buffer that

Appendix C: Use of quantitative DNA ladder 2014
provides ions to carry a current and some type of buffer to maintain the pH at a
relatively constant value.
Agarose
is
a
polysaccharide
extracted from seaweed. Agarose gels
are prepared by mixing agarose powder
with buffer solution to a final
concentration of 0.5 to 2%, followed by
heating until a clear solution is obtained.
Most commonly, ethidium bromide (final
concentration 0.5 ?g/ml) is added to the
gel at this point to facilitate visualization
of DNA after electrophoresis. However,
as ethidium bromide is a toxic mutagen,
we will use a safer DNA stain instead,
known as SYBR safe® (Invitrogen). After
cooling the solution to about 55°C, it is
poured into a casting tray containing a
sample comb and allowed to solidify at
room temperature or in the cold. The
porosity of the gel is inversely related to
the agarose concentration. By varying
the concentration of agarose, fragments of DNA from about 200 to 50,000 bp can be
separated. The higher the agarose concentration, the "stiffer" the gel and the smaller
the size of the DNA or RNA fragments that can be separated.
Following separation, DNA fragments are visualized by staining with SYBR-safe.
This fluorescent dye intercalates between bases of DNA and RNA. It is often
incorporated into the gel so that staining occurs during electrophoresis, but the gel can
also be stained after electrophoresis by soaking in a dilute solution of SYBR-safe. DNA
or RNA bands appears in red-orange color when the gel is exposed to UV light.
Fragments of linear DNA migrate through agarose gels with a mobility that is
inversely proportional to the log of their molecular weight. Circular forms of DNA
migrate in agarose differently from linear DNAs of the same mass. Several factors have
important effects on the mobility of DNA fragments in agarose gels, and can be used to
advantage in optimizing separation of DNA fragments. Among these factors are:
agarose concentration, voltage (as the voltage applied to a gel is increased, larger
fragments migrate proportionally faster than small fragments), electrophoresis buffer
and SYBR-safe (when present in the gel). The molecular weight of a linear DNA sample
can be determined by running a mixture of linear DNA fragments of known size under
the same conditions (Figure 3).
Figure 3: MassRuler Express Forward DNA ladder marker. The MassRuler Express
Forward DNA ladder is constituted of 12 DNA fragment varying in size from 100 base
pairs (bp) to 10,000 bp. Predetermined quantity of each fragment were mixed to

Appendix C: Use of quantitative DNA ladder 2014
produce this marker. These quantities are dependent on the volume of marker to be
loaded on the agarose gel. For example, 5 microlitres (?L) of the marker contains 50 ng
of the 1000 bp fragment whereas 10 ?L of the same marker will contains 100ng of the
same fragment. It is important to remember that linear DNA fragments migrate through
agarose gels with a mobility that is inversely proportional to the log of their
molecular weight
H. Restriction Endonuclease Mapping of a Plasmid
Restriction endonucleases are a family of site-specific endonucleases that cleave
the phosphodiester bond in the phosphate back bone of DNA. This cleavage is a
hydrolysis reaction, adding a molecule of water across the phosphodiester bond,
breaking it and generating a 5’ phosphate and a 3’ hydroxyl end (Figure 4A). These
enzymes recognize short (4-8bp), often palindromic (identical when read 5’ to 3’ on
each strand) sequences. The cleavage of both strands of the DNA backbone generates
either blunt or sticky ends, depending on the specific enzyme used (Figure 4B). These
ends are exploited for many molecular biology used to manipulate DNA, most notably
cloning.
When plasmid DNA is isolated and run on an agarose gel, several bands may be
seen. The predominant form a plasmid takes is a supercoil, often likened to an over
twisted telephone cord, the double helix twists on itself yielding a compact piece of DNA
that migrates faster than its linear form. A plasmid may be damaged or in the process
of being replicated at the time of isolation resulting in a nicked formation, likened to a
big floppy circle, being adopted. The nicked format migrates slowly mimicking a DNA
fragment longer than its linear form. Finally, during alkaline lysis it is possible to use
overly harsh conditions resulting in a permanently denatured intact single strand of the
plasmid, this form migrates the fastest on an agarose gel (Figure 4C). Following a
restriction digest it is often helpful to run both a digested aliquot and an undigested
control on an agarose gel to confirm that the digestion is complete based on the
banding pattern observed.
With each restriction enzyme having a unique recognition sequence, each time an
identical piece of DNA is digested with the same enzyme, a characteristic fragmentation
pattern will be observed. Exploiting this reproducibility, restriction mapping consists of
digesting DNA with a series of restriction enzymes and separating them on an agarose
gel to visualize the resulting fragments (Figure 4C). These fragmentation patterns are
then be used to characterize the relative location of the restriction sites be it on an
unknown piece of DNA, as will be done in this experiment or to track and confirm the
results of manipulating DNA (such as when inserting of a gene of interest in to a
plasmid).

Appendix C: Use of quantitative DNA ladder 2014

Figure 4. Restriction digestion and plasmid mapping. A) Adding a molecule of
water across the phosphodiester bond breaks the phosphate backbone of the DNA,
generating a 5’ phosphate and a 3’ hydroxyl end on the resultant DNA fragments (figure
modified from http://chem wiki.ucdavis.edu) B) Digestion with restriction enzymes may
result in sticky ends, as seen for HindIII and EcoRI or blunt ends, as seen with EcoRV.
C) A schematic depicting the restriction digest of a plasmid using two enzymes: where a
single digest with each enzyme and a double digest were completed and compared with
the undigested plasmid, in all its possible conformations and a molecular weight
marker.

Appendix C: Use of quantitative DNA ladder 2014

Appendix C: Use of quantitative DNA ladder 2014
PROCEDURES
In experiment 1 you will extract the E. coli DNA. For experiment 2, a stock solution
of E. coli DNA from a commercial source will be provided by the lab technician so that
you can start the first “melting” curves (step 16) early in the lab session. In this
experiment, you will analyze the effect of salt and a denaturing agent on DNA structure.
Using the same “melting” technique, you will compare the homogeneity and integrity of
the in-lab prepared DNA with a commercial source of E. coli DNA. In experiments 3 and
4 you will be introduced to the techniques of restriction digestion and DNA
electrophoresis on agarose.
Before performing these experiments, you should watch the following videos:
? Gel electrophoresis Part 1 (13:05) or (http://www.youtube.com/watch?
v=3ukaT_Ih9d8)
? Gel electrophoresis Part 2 (8:13) or (http://www.youtube.com/watch?
v=_QxxB65Gi78)
EXPERIMENT #1: Isolation and characterization of bacterial DNA
a)DNA extraction

1. You are provided with 60 mg of Escherichia coli, strain B, (a gram negative
bacterium) suspended in 4 mL of cold Standard saline-EDTA (0.15 M NaCl in 0.1
M ethylene-diamine tetra-acetate – EDTA, pH 8.0) in a 50 mL Falcon tube.

2. Add 375 ?L of 25% sodium dodecyl sulfate (SDS) and mix gently by inversion.
(SDS is a detergent and will foam extensively; you could also easily shear the
DNA).
CAUTION! The SDS solution may irritate the skin and eyes. Use gloves and
safety glasses.
(http://ccinfoweb2.ccohs.ca/msds/Action.lasso?-database=msds&layout=Display&-response=detail.html&op=eq&MSDS+RECORD+NUMBER=5502925&-search )

3. Incubate the mixture in a 60°C water bath for 10 minutes and then cool to room
temperature.

Appendix C: Use of quantitative DNA ladder 2014

4. Add 0.725 mL of 6.0 M NaClO4 and mix gently.
CAUTION! The perchlorate solution may irritate the skin and eyes. Use gloves
and safety glasses. (http://www.sciencelab.com/msds.php?msdsId=9925018 )

5. Add 5.0 mL of chloroform:isoamyl alcohol (24:1, v/v) in the fume hood.
CAUTION! Chloroform and isoamyl alcohol are toxic and irritate skin, eyes and
respiratory tract. Keep away from sparks and flame. Use gloves and safety
glasses. Avoid inhalation. Work in the fume hood.
(http://www.sciencelab.com/msds.php?msdsId=9927133 )
(http://www.sciencelab.com/msds.php?msdsId=9927550 )

6. Mix gently on a waver for 5 minutes.

7. Centrifuge at 12,000 xg for 5 minutes. (Tubes should be balanced!).

8. Carefully remove 70% of the upper aqueous phase by aspirating the liquid off
with a plastic pipette and transfer it to a 15 mL Falcon tube. Avoid contaminating
your sample with the denatured protein that collects at the interface between the
aqueous and organic phases. Dispose of the chloroform phase in the appropriate
waste container.

9. Gently layer 10 mL of 70% ethanol over the aqueous phase in the tube. Seal the
tube with its cap and mix gently and continuously until the ethanol becomes fully
mixed with the aqueous phase (Observe the precipitation of the DNA).

10. Retrieve the DNA using a glass rod and immerse it into a microtube containing 1
mL of 70% EtOH. Mix it gently in this solution to remove the salts.

Appendix C: Use of quantitative DNA ladder 2014
11. Squeeze out as much liquid as possible from the spooled mass by lightly
pressing the stirring rod against the side of the tube. Place the glass rod in an
upright position and allow it to drain for at least 10 min. Failure to remove the
alcohol effectively will lead to difficulties in dissolving the DNA in the next step.
Further removal of ethanol can be achieved with a stream of air.

12. Dissolve a fraction of the crude DNA on the glass rod by stirring the DNA into 2
mL of 15 mM citrate buffer (sonicated), pH 7.0 in a 15 mL centrifuge tube. Gently
swirl the glass rod back and forth until the DNA becomes detached from the rod.
Seal the tube and place it on a waver for 5 min.

13. Transfer 1 mL to a 1.5 mL microtube and centrifuge it for 1 min at 13,000 rpm.

b) DNA characterization
You will determine your DNA yield as well as the degree of purity and integrity of a
DNA sample prepared as above.

14. Pipette 200 ?L of your dissolved DNA from step 13 into a quartz cuvette
containing 800?L of 15 mM citrate buffer. Mix by inversion. Read and record the
absorbance at 234, 260 and 280 nm of your sample with the UVspectrophotometer, using the 15 mM citrate buffer as the blank.

15. Prepare a dilution of your DNA sample with a final volume of 1mL in 15 mM
citrate buffer, pH 7.0 in order to have an A260 between 0.4 and 0.5. Calculate and
record your dilution factor. This solution will be used for the DNA melting curve
experiment (step 18).
EXPERIMENT #2: DNA melting curves
For a melting experiment you will follow the change in absorbance at 260 nm of
the DNA in function of the temperature. You will use the Cary100 Bio system that can
analyse 12 DNA samples simultaneously.
Melting experiments will be performed on the following DNA samples:

Appendix C: Use of quantitative DNA ladder 2014
a) E. coli DNA from a commercial source,
b) E. coli DNA from a commercial source, in the presence of salt,
c) E. coli DNA from a commercial source, in the presence of dimethyl
formamide,
d) E. coli DNA from step 15.
Each team will perform one of the three meltings a-c plus its own sample (d).

16. Prepare 1 mL of a 1:20 dilution from the stock commercial DNA solution as
indicated below: (Take 50 ?L of the stock solution and add it to 950 ?L of the
corresponding buffer).
Note: All buffers have been de-aerated by sonication. This will prevent bubble
formation during the heating process.

solution a: commercial E. coli DNA in 15 mM citrate buffer, pH 7
(Teams 1, 2, 3, 8, 9, 13, 14, 15, 17, 19, 20 and 21)
solution b: commercial E. coli DNA in 15 mM citrate, 0.10M NaCl
(Teams 4, 5, 10, 11, 22 and 23)
solution c: commercial E. coli DNA in 15 mM citrate, pH 7, 25% DMF
(Teams 6, 7, 12, 16,18 and 24)

CAUTION! Dimethyl formamide (DMF) solutions may irritate skin and eyes. If
absorbed, it may harm the unborn child (teratogen). Use gloves and safety
glasses.
(http://ccinfoweb2.ccohs.ca/msds/Action.lasso?-database=msds&layout=Display&-response=detail.html&op=eq&MSDS+RECORD+NUMBER=3627195&-search )
17. Once samples from all teams are loaded into the Cary 100Bio spectrophotomer
(as directed by your TA), the technician will start the melting experiment.

Appendix C: Use of quantitative DNA ladder 2014
18. Once experiment 1 is completed, each team will set up a second melting
experiment with your own extracted DNA sample. Samples from teams 1 to 12
will be run in one apparatus and samples from teams 13 to 24 in the second
apparatus. Please record the cell number in which your sample is loaded.
19. Once the melting is finished, you will see the two plots of the absorbance and the
1st derivative on the same graphic. The out-put from the instrument (BTM file) will
be saved and posted on the course’s web site in the Virtual Campus as a CSV
file. (Excel can open this file and save it as a XLS file which is the Excel format).
For convenience, the files will be named in the following format: L5S12(112)abc.csv or L5S12(1-12)d.csv; where S12 is the second Thursday afternoon
section and (1-12) indicate samples from teams 1 to 12. For your report you will
need the results corresponding to your sample plus any set of the three
commercial samples in different conditions (control, salt and DMF) that were
analyzed the same day. You are to incorporate the four graphics (a - d) in your
report showing the two plots for the absorbance and the 1 st derivative.
EXPERIMENT #3: Restriction Endonuclease Mapping of a Plasmid
In this experiment, an unknown plasmid (100 ng/µL) will be digested with a
combination of two different restriction enzymes: EcoRI (10 U/µL) and HindIII (10 U/µL).
You will be supplied with H2O, a solution of 10X digestion buffer (Buffer O) and an
unknown plasmid (100 ng/µL). Each team will be digesting a different plasmid.
20. Prepare the following reaction mixtures in 4 labelled 0.5 mL microcentrifuge
tubes by adding the following reagents in the order they are listed. Ask your TA
for the EcoRI and HindIII restriction enzymes, adding them to the mixture last;
rinse the pipette tip with the solution at least 5 times. Always keep your tubes on
ice until loading it into the waterbath.

Appendix C: Use of quantitative DNA ladder 2014
Table 1. Plasmid digestion mixture

Reagents

EcoRI

HindIII

H2O (brown tube)
10X Buffer (blue tube)
Unknown plasmid (100
ng/µL)
(Orange tube)
EcoRI (10 U/µL) (see TA)
HindIII (10 U/µL) (see TA)
Total volume

11 µL
2 µL

11 µL
2 µL

EcoRI +
HindIII
10 µL
2 µL

6 µL

6 µL

6 µL

6 µL

1 µL
---20 µL

---1 µL
20 µL

1 µL
1 µL
20 µL

------20 µL

Control
12 µL
2 µL

21. Mix the solutions by tapping the bottom of the tubes with your finger. To bring all
the reagents to the bottom of the tube, centrifuge for 15 seconds. (Make sure
your tubes are balanced!)
22. Incubate your tubes in the 370C water bath for 1 hour.
23. Once the digestion is completed, add 2.2 µL of 10X DNA-electrophoresis loading
buffer to each sample. Mix thoroughly.
Proceed now with electrophoresis on the provided gel (step 29).
EXPERIMENT #4: Agarose gel electrophoresis

N.B. To avoid waiting time, a gel for every four groups is provided. Your TA will
demonstrate how to prepare a gel (steps 24-28). Groups of 4 teams will load a gel
and perform the electrophoresis as described in steps 29-34. Before loading the
sample, you can practice with loading buffer and the demo gel.

24. In a 250 mL bottle, prepare 100 ml of a 1% (w/v) agarose solution in TAE (40 mM
tris-acetate, 1.0 mM EDTA) buffer. Check calculation with your TA before
proceeding.

25. Heat the agarose suspension in a microwave oven. To prevent pressure from
building up inside the bottle, loosen the cap before heating. Use high setting 3 x
1 min, mixing after each minute. Make sure agarose is completely melted.

Appendix C: Use of quantitative DNA ladder 2014
26. Once dissolved, allow to cool to 50-55°C (agarose solidifies at about 40°C); any
hotter than 55°C will melt the gel casting mold.

27. When the gel solution is at 50-55°C, add 10 µL of SyBr Safe DNA Stain.

28. Place the gel running tray in the gel casting mold and pour the cooled (50-55°C)
agarose solution into the casting mold. Pour slowly to avoid making bubbles.
Place a 20 well comb at 1 cm from one end of the casting tray. Dislodge any
bubbles with a pipet tip. Allow the gel to solidify for at least 20 min.

29. Remove the comb by slowly lifting one end, making sure not to suck the agarose
out of the bottom of the well.

30. Lift the gel tray from the casting mold and place in the electrophoresis chamber.
Pour in the TAE buffer until the gel is covered by 4-8 mm of buffer.

31. Load 10 µL of provided MassRuler Express Forward DNA ladder marker (M) and
15 µL of the samples from step 23 in wells 1 to 20 as described in the template
bellow. Place the end of the tip below the surface of the running buffer
immediately above the desired well. Expel the sample slowly (taking care not to
allow any air bubbles to form under the tip), the high concentration of glycerol in
the loading buffer will cause the entire sample to sink to the bottom of the well.

Gel loading template

Appendix C: Use of quantitative DNA ladder 2014

32. Place the lid on the electrophoresis unit and connect the electrodes such that
migration proceeds towards the RED (positive, anode) electrode. Connect the
leads to the power supply and apply 100 V for 45 min.

33. Turn the power supply off, disconnect both ends of the electrodes and remove
the gel tray. (WEAR GLOVES)

34. Place the gel and running tray on the UV Gel Doc System and take a picture.
(Ask your TA for help). For convenience, name the file in a similar way as for the
melting data by giving the lab number, the section and the number of the teams
with samples on the gel (e.g. L5S2(5-8)).

Appendix C: Use of quantitative DNA ladder 2014
RESULTS AND DISCUSSION

Appendix C: Use of quantitative DNA ladder 2014
Quantitative DNA Ladder and estimation of length and amount of DNA bands
A quantitative DNA ladder contains known amounts of DNA molecules of different
lengths. It serves as a reference to estimate the size and amount of an unknown DNA
band. A quantitative DNA ladder must be used each time you are loading sample on
and agarose gel; it must be loaded at the same time as your samples.
Length of an unknown
One of the first information you can draw from the use of a DNA ladder is the
estimation of the length of an unknown DNA band. By quickly comparing the length of
your unknown band to the bands of the molecular ladder, you can estimate its length
(Figure 1A). If you want to be more precise, one can a plot describing the log(length) vs
mobility for the different bands of a marker, and then use the equation derived from the
trendline to predict the length of an unknown. Here is how you can achieve that:
a) Measure the travelling distance of each band of the marker by using a ruler (from
the well to the middle of each DNA band). See Figure 1B.
b) Plot the log of the length of the DNA fragments against their travelling distances
(Scatter plot in Microsoft Excel). See Figure 1C.
c) Create a linear regression line (Linear trendline option in Excel and check the
Display Equation on chart option. See Figure 1C.
d) You can now measure the travelling distance of your unknown and input this
value in the linear regression equation to get a prediction of the length of your
unknown sample.
As you can see in the presented example, high molecular weight fragments
sometimes affect the results of the linear regression. Large DNA molecules (10,000bp
+) have a really hard time migrating through the molecular mesh of a 1% agarose gel
and therefore, it becomes more difficult to measure the migration distance of these
fragments with precision. If you notice outlier values on your linear regression line, you
can eliminate some of them to improve your R 2 as I did for the example presented.
Amount of an unknown
You can also quantify (estimate) the amount of DNA that was loaded on gel by
direct comparison to the DNA ladder. The intensity of a band is proportional to the
number of SYBR Safe molecules bound onto the DNA and therefore, this intensity will
vary with the number of bp and the amount of the DNA fragment that was loaded on gel.
There are two rules to this type of estimation, first, always try to use a band of the
marker that is closely related in size to your unknown since larger fragments will
intercalate more dye than small fragments, which in turn, will give them greater band
intensity. Secondly, once you have selected a band of the marker for your comparison,
use the information given by the manufacturer (amounts of DNA by loading volume) to
know how much DNA is present in that band. Knowing the amount of DNA present in

Appendix C: Use of quantitative DNA ladder 2014
the marker band (from the manufacturer information sheet), you can then apply the
intensity ratio to this number. For example, if you look at gel presented in Figure 1A,
the unknown band is 3 times more intense that the 3000 bp marker band. If 30 ng of this
DNA fragment was loaded on gel (10 ?L of the marker), I can say that I have loaded
~90 ng of my unknown.

Figure 1. Quantitative DNA Ladder and estimation of length and amount of DNA
bands. A) Estimation of a DNA fragments length using the MassRuler Express Forward
marker (Fermentas). DNA quantity presented on the right side of the marker description
sheet correspond to amounts of DNA loaded for each fragment of the marker in relation
to the volume of marker used (top line). B) Determination of the DNA fragments length
by plotting the logarithm of the length of the DNA fragments of the marker against their
migration distances. C) Linear regression of the log (fragments length) against the

Appendix C: Use of quantitative DNA ladder 2014
migration distance. Points located at the end of the regression line were eliminated to
improve the R2 value of the line.

Appendix C: Use of quantitative DNA ladder 2014

Appendix D: Supplementary figures 2014

Appendix D: Supplementary figures 2014

Lab 3 Supplementary Figure 1. Enzyme inhibition effects observed using
Lineweaver-Burk (LWB) representation. A) Michaelis-Menten plot of v0 against
increasing [S], where vo approaches Vmax at high [S] and half of this maximum velocity
corresponds to KM. B) Taking the reciprocal of the Michaelis-Menten equation and
plotting 1/v against 1/[S] allows for analysis by linear regression, where the slope =
KM/Vmax, y-intercept = 1/Vmax and x-intercept = -1/KM. In the case of COMPETITIVE
inhibition, Vmax is unaffected but the amount of substrate needed to achieve it increases.
This is graphically visible by a change in the shape of the curve for the MichaelisMenten plot due the increase in Km by factor of ?; where : ? = 1 + [I]/KI (C) and an
increase in the slope ((?Km/Vmax) of the LWB plot along with an increased x-intercept (1/?Km) (D). In the case of UNCOMPETITIVE inhibition the both the Km and Vmax are
decreased by a factor of ?’, where ?’ = 1 + [I]/KI’. This results in an overall lowering of
the Michaelis-Menten curve (E) and an upward shift of the LWB plot. While the slope of
the LWB remains unchanged, the y-intercept (?’/Vmax) increases and the x-intercept
decreases ((-?’/Km) (F). Finally in the case of mixed inhibition the V max decreases
(Vmax /?’) while the Km increases (?Km/?’???This is observed as a lowering and flattening
of the Michaelis-Menten plot (G) and an increase in the slope (?Km/Vmax) of the LWB plot
due to the increased y-intercept (?’/V max) and decreasing x-intercept (-?’/?Km)(H).

Lab 3 Supplementary Figure 2. Enzyme inhibition effects observed using the
Hanes representation. A) Michaelis-Menten plot of v0 versus increasing [S], where vo
approaches Vmax at high [S] and half of this maximum velocity corresponds to K M. B)
The Hanes plot is an alternative linear model that can be obtained by multiplying the
Lineweaver-Burk equation by [S] and plotting [S]/v against [S]. In this linear-regression,
the slope corresponds to 1/V max, y-intercept to KM/Vmax and x-intercept to -KM. In the case
of COMPETITIVE inhibition, Vmax is unaffected but the amount of substrate needed to
achieve it increases. This is graphically visible by a change in the shape of the curve
for the Michaelis-Menten plot due the increase in K m by factor of ?; where : ? = 1 + [I]/KI
(C). The slope of the Hanes plot remains unchanged, while the y-intercept (?Km/Vmax)
increases and the x-intercept decreases (-?Km). (D). In the case of UNCOMPETITIVE
inhibition the both the Km and Vmax are decreased by a factor of ?’; where ? = 1 + [I]/KI’.
This results in an overall lowering of the curve in the case of the Michaelis-Menten plot
(E) an increase in the slope (?’/Vmax) of the Hanes plot due to an increased x-intercept (Km/?’) (F). Finally in the case of mixed inhibition the V max decreases (Vmax /?’) while the
Km increases (?Km/?’???This is ovserved as a lowering and flattening of the MichaelisMenten plot (G) and an increase in the slope (?’/Vmax) of the Hanes plot due to the
increased y-intercept (?Km/Vmax) and decreasing x-intercept (-?Km/?’)(H).

Appendix D: Supplementary figures 2014