CHE 352 – Chemical Engineering Laboratory I
Pre-Lab Instructions
Pre-Lab Preparation
Prior to receiving authorization from the TA/Instructor to begin an experiment, teams
must work together to collectively prepare a satisfactory pre-lab report. Although the
entire group is responsible for helping to prepare the content of the pre-lab, one
student will serve as the Project Leader (the Leader position will rotate every
experiment). The Leader will serve as the primary presenter of the pre-lab to the TA
or Instructor however, all students must be in attendance during the presentation and
should be aware of the entire experimental plan and all pre-lab content.
The TAs will review pre-labs at the start of the lab and will quiz ALL team members
before granting approval to begin experiments. We will not permit teams to begin
unless sufficient preparation has been done! The name of the Leader should be
clearly indicated on the Pre-lab document.
Note: Be aware that not all the information that you will need to perform the lab and
eventually write the report is available in the hand-outs provided by the instructor! This
was done intentionally. One critical component of this course is to develop students’
abilities to look up and gain understanding of necessary information by themselves.
Another important component of this course is to develop the students’ ability to
properly design and plan experiments. Detailed, step-by-step instructions are not
provided for the labs; it is up to the student to think about and develop their
approach. The prelab should illustrate satisfactory preparation in all these areas.
Pre-Lab Submission
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Pre-Labs should be clear/concise and a maximum of 2 pages in length (use of
bullet points is encouraged – be efficient!).
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All team members must contribute to the preparation of each Pre-Lab (Pre-Labs
are graded as a team!)
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Project Leaders must ensure the Pre-Lab is uploaded to Canvas prior to the first
session of a new experiment. Students will not be allowed to start an experiment
without a completed pre-lab submitted and penalties will be applied for late
submissions.
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The pre-lab must address the following essential areas of importance:
1. Goals and Hypothesis – what are the goals of and/or central questions that you
intend to answer/address/explore in the experiment? Support this with background
information, as appropriate.
a. What is your hypothesis for your experiments? What do you think will
happen?
b. Your proposed goals and ultimately the work presented in your report must
demonstrate a reasonable amount of effort (i.e., use your time in the lab
wisely and to its fullest potential).
2. Experimental Approach – describe your experimental protocol in stepwise
manner. What will be measured when and why? Most importantly, indicate why
the experiments and acquired data are essential to addressing the central
hypothesis.
a. Your experimental plan should account for the amount of time allotted for
each experiment (5h 40min total) and include a rough schedule.
3. Analytical Approach – how will the raw data be analyzed and why? Provide
examples of key equations and the anticipated workflow for data analysis (e.g.,
‘we will first calculate ‘x’ using the ‘y’ equation before comparing it to ‘z’, and so
on).
a. Don’t simply list equations here – explain how they will be used
4. Error and Calibrations – describe how your team will perform error analysis (e.g.,
error propagation or replicate experiments? If replicates, how many? Which
ones?). How will you ensure that your measurement devices are as accurate as
possible? Describe all relevant experimental controls and/or calibrations that you
will perform and why these are important.
a. Many of the experiments require operation at steady-state – how will
you ensure that you are operating at steady-state for your specific
experiment?
5. Workflow and Management – what roles will each of the team members be
playing and how will the efforts be effectively coordinated?
6. Safety – what are the relevant safety concerns associated with the experiment and
what specific steps will you follow should an accident occur? (note: please omit
standard things like ‘wear goggles’, etc., focus on unique and specific
hazardous associated with the experiment in question).
Please note, references are not necessary for pre-labs but you can feel free to include
them if you would like.
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CHE 352
Electrophoresis
Make sure you are using latex or nitrile gloves for all parts of this experiment!
Casting a Gel:
1. You should fill each gel tray with ~11 mL of heated gel mixture
2. Gel mixture includes:
a. DI water (what does DI stand for?)
b. 10x TBE (or TAE) buffer (how much should you add?)
c. Agarose I (how much to add for a 1% w/v gel? What about 2%?)
d. Check these numbers with a TA before starting your lab!
3. Heat gel mixture on hot plate until agarose is dissolved
4. Pour gel mixture into the gel tray in the casting stand (Make sure to add combs!)
5. Let cool until hardened
Loading a Gel:
1. Place gel tank into the carriage and ensure that black viewing platform is in the gel tank
2. Place gel tray (with cast gel!) into the gel tank on top of the black viewing platform
a. Which way should the gel tray be oriented? Which side should the wells be on?
Which direction does DNA travel?
3. Add ~135 mL of 1x TBE (or TAE) buffer to the gel tank
a. What does 1x mean? How to make 1x buffer?
4. Add DNA samples into wells (~10 uL of DNA sample per well; you probably will only
need to use 1 or 2 wells per gel)
a. The DNA has already had dye added to it so that it can be visualized
b. Make sure to check with a TA before you do this!
c. Do put your pipet tip too deep into the well! If you puncture the gel, your DNA
sample will fall out of the bottom!
5. Place orange photo hood on the carriage
6. Press the power button (only if it is now a solid green light)
a. If it’s not green, something is wrong!
7. Turn on low (or high) intensity blue light (don’t forget to start your timer!)
8. Do not let DNA bands run off the gel – turn off power before this occurs
Preparing DNA samples:
1. If DNA samples do not have loading dye, you will need to add it to the samples
a. How much to add?
Clean-Up:
1. At the end of lab, pour used running buffer (TBE buffer) into the TBE buffer container
(this can be reused)
2. Throw gel into a bio-waste bag/bin
a. Make sure to take a picture of your gel before you throw it away
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CHE 352
Electrophoresis
Broad Objectives: To study forced mobility of charged molecules using electrophoresis.
Background: Migration of charged particles such as DNA and RNA through a solution under the
influence of an applied electric field usually provided by immersed electrodes is called
electrophoresis. DNA with negative charge moves toward the anode, which is a positively
charged electrode. Notably, the determination of the human genome has been attributed to
Electrophoresis.
Experimental Notes: You will be provided the following equipment:
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1 x MiniOne carriage with blue LED lights
1 x buffer tank with graphite electrodes
1 x casting system with 2 gel trays and 2 reversible combs for 6 and 9 well gels
1 x amber filter photo hood for real-time viewing and capturing gel images
1 x 42V (output) power supply
1 x 2–20µl Variable Volume micropipette
10x TBE buffer concentrate
Agarose I
Deionized (DI) water
New England BioLabs (NEB) 1 Kb DNA Ladder (i.e., DNA sample)
(https://www.neb.com/products/n3232-1-kb-dna-ladder#Product%20Information)
-See Figure 1 below for picture/details of the DNA ladder
-Note: the DNA sample has already had dye added to it so that it can be visualized
Figure 1. DNA bands from NEB 1 Kb DNA ladder shown with corresponding DNA length (in number of
kilobases) and DNA mass (in ng).
Gels will be made of agarose, a polymer of carbohydrates. Gels will be formed within a range of
0.6 to 1.5% agar (w/v) (this will be something that you change over several runs), carefully
melted in a microwave oven (or hot plate) before casting. Lower concentration gels lead to
DNA running faster. You should test out at least 3 different agar concentrations and one DNA
sample should be run for each gel (if you think your first DNA sample will not provide good
results, you can add a second sample).
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CHE 352
A comb should be used to generate wells in the gel for loading DNA solutions. A good design
has teeth along the long edge of the gel. Allow the comb to be vertically positioned just above
the surface of the casting tray. The wells should be as deep as possible, but several millimeters
off the bottom of the gel to allow for the wells to be formed. The cooled gels can now be lifted
out of the tray and placed in the gel running container.
The agarose gel electrophoresis system utilized here will use a solution called “running buffer”
(in this case, TBE buffer). It is critical to buffer the pH so that DNA is not degraded. It is
expected that the team members will come to the lab knowing the recipe to generate buffers
(i.e., teams are provided a concentrated TBE buffer solution as shown in the list above; how will
you dilute it?).
One of the most significant issues that students run into in this lab are pipetting issues. Please
be sure you know how to utilize a pipette; Figure 2 gives an illustration of how to properly use a
pipette to move liquids from one container to another. Additionally, the following video gets
some instructions on how to effectively load DNA into gels – please watch, as this is one of the
more difficult technical aspects of this experiment (Gel Electrophoresis Loading Video – most
useful info begins at 0:45).
Figure 2. Illustrations and instructions on how to properly pipette liquids. Be sure to be aware that for
pipettes, there are two stops when plunging up and down. The first stop is used when you are taking up
(aspirating) liquid; the second stop is used when you are dispensing liquid. One thing not mentioned in
the graphic above is setting your selected volume. Make sure you are using the correct pipette for the
your desired volume (see pipette for volume range) and that you have set the pipette to the correct
volume (ask TA/instructor for help if you are unsure) (source: https://slideplayer.com/slide/12844569/).
Pre-lab requirement: You must come to lab with a prepared recipe for a 1% gel. You must
know what volume to make (each gel tray has a minimum volume 11 mL, how much should you
make?), and how much DI water/concentrated TBE buffer/SYBR Safe and Agarose to add (See
Electrophoresis Cheat Sheet in Canvas for more information).
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CHE 352
Theory and Background:
In this experiment we are going to measure the rate of migration of DNA in the gel and the
effective radius of the DNA.
Recalling fluid dynamics principles, the drag force of a particle moving through a fluid is
proportional to velocity (from Stokes’ Law). Therefore, at some critical velocity, the electric force
on the DNA will be equal to the drag force on the DNA (what would happen if there was no
drag force?). What does this tell us about the velocity of the DNA at this point? Is the velocity
when the electric force is equal to the drag force the same for DNA of all sizes? Equations
representing the electric (Eq 1) and drag force (Eq 2) on DNA are shown below:
???????? ????? ?? ??? = ?? = ??
(1)
???? ????? ?? ??? = ?? = ?∙? = 6∙π∙ η ∙??∙?
(2)
By combining these equations, we can find an equation (Eq 3) to represent the mobility of DNA,
as shown below:
µ =
?
6∙ π ∙ η ∙??
=
?
?
(3)
f = frictional coefficient
μ = mobility
q = charge of the DNA
η = effective viscosity of the gel matrix (this is your dependent variable – this will change
depending on concentration of gel AND DNA size – you must explain why this occurs!)
RH = hydrodynamic radius of the DNA (what are we assuming about the DNA here?)
ν = velocity of the DNA molecule
E = Electric field = Voltage/Length (between electrodes)
DNA is a polymer and as such can be represented by a radius of gyration (RG) and
hydrodynamic radius (RH), which can be estimated with Eq 4 and Eq 5, respectively:
−10
?? =
2∙?∙?∙(3.4*10
6
?? = 0. 662??
−8
P = Persistence length of the DNA polymer = 5 * 10
N = Number of base pairs in the DNA
?)
(4)
(5)
?
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CHE 352
There are other models to estimate the radius of gyration and hydrodynamic radius of DNA, feel
free to use whichever one you like (cite your source) and remember that no model is perfect.
Minimum Analysis:
1. Discuss the properties of the agarose gel and how they affect the transport of DNA
through the gel. Is DNA flowing through an agarose gel similar to DNA flowing through a
liquid (e.g., TBE buffer)? What are the important mass transfer characteristics of the
gel?
2. Utilize 3 – 5 DNA lengths to compare and illustrate how mobility and effective viscosity
change with various properties.
a. Show graphs of mobility, hydrodynamic radius of the DNA and viscosity related to
DNA length in your report!
b. Be sure to include pictures of some of your gels for reference
3. What can you determine about mobility and effective viscosity of different sizes of DNA
within a single gel concentration?
4. What can you determine about mobility, effective viscosity, and hydrodynamic radii of the
same size DNA among different gel concentrations?
5. If you had a sample to run on a gel that you think should contain bands of 700, 800 and
900 bp in length, which gel concentration (of those tested) would you use to achieve the
best separation? Utilize your data along with theory and literature to provide evidence for
your answer!
6. In this lab, the electrophoresis apparatus provided a constant voltage (42 V) to your gel.
However, in many electrophoresis applications, this voltage is variable and can be
changed depending on the application.
Utilizing your new understanding of
electrophoresis theory, how do you think changing voltage affects DNA separation and
transport in the gel? Feel free to utilize sources for this discussion!
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