Lab 3

Optimization of Transfection Using

CaPO4 & Lipofectamine 2000 Methods

Animal Cell Biology

BIOT 4230
Winter, 2013

3.1: TRANSFECTION
Background
Gene transfer into animal cells is a very common lab technique but there are many
different procedures used to accomplish this task. Introduction of DNA (typically
plasmid vectors) into cells is termed transfection. The most common transfection
methods involve the formation of chemical complexes of DNA with a variety of
reagents, the most common being CaPO4, polyethylenimine (PEI), lipids (e.g.
Lipofectamine 2000) or dendrimers (e.g. Superfect).
In this experiment, you will be comparing lipid-based versus CaPO4 transfection to
determine which method is more effective (in your hands) in delivering a plasmid
(see Figure 1), which carries the GFP reporter gene, into NIH-3T3 cells.
Additionally, you will be varying the amount of plasmid DNA (and lipid) to optimize
transfection and the consequent expression of GFP.

Figure 3.1. GFP Reporter Plasmid

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Materials and Reagents
NIH-3T3 Complete Medium (pre-warmed)
6-well tissue culture-treated plates (2 plates per pair of students)
Sterile plasmid DNA (0.5 g/mL)
Sterile cell culture-grade H2O
Sterile microfuge tubes
Set of Gilson pipettes & sterile tips
CaPO4 Method:
2X HEPES buffered saline
2.5 M CaCl2
Lipofectamine 2000 Method:
Lipofectamine 2000 (Invitrogen)
DMEM (no serum, no antibiotics; see Lab 1))

3.1.1 Seeding Cells for Transfection

Procedure
On the day before transfection, seed two 6-well plates as follows:
1.

Inspect the NIH-3T3 flasks you have been maintaining over the past 2 weeks
and select 2 or 3 of your most confluent T25 flasks that have been in continuous
logarithmic growth
Guidelines: you will need 5 x 106 total cells for seeding and one 95% confluent
T25 flask contains ~2.5 to 3 x 106 total cells

2.

Trypsininze and block all of your chosen flasks to generate a single-cell

suspension (SCS) as follows:
a. Follow the trypsinization procedure described in Lab 2, except pool your
SCS (from each flask) into a sterile conical test tube
b. Keep the final volume of your pooled single cell suspension between 20 to
24 mL
c. If you are harvesting from 2 flasks, block with 8 mL of Complete Medium and
pool the 2 x 10 mL SCS for a total volume of 20 mL
d. If you are harvesting from 3 flasks, block with 6 mL of Complete Medium and
pool the 3 x 8 mL SCS for a total volume of 24 mL

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3.

Count an aliquot of your pooled SCS using Trypan Blue and hemacytometer (as
per Supplementary protocol, Cell density and viability assessment) and
determine the viable cell density (VCD = cells/ml) and viability of the SCS.

4.

Adjust the VCD of your pooled SCS by diluting with Complete Medium
according to the following specifications:
a. seed 4x105 total cells into each well of a 6-well plate using a seeding
(delivery) volume of 2 mL per well
b. Seed 2 x 6-well plates = 12 wells total

5.

Invert your (capped!) tube of SCS (adjusted to the desired density) several times
to resuspend the cells, then use a 10 mL pipette to seed 4x105 cells into all wells
of two 6 well plates using a seeding volume of 2 mL/well.
Note: you will be using 1 plate for the Lipofectamine 2000 transfection and 1
plate for the CaPO4 transfection

6.

Incubate plates in 37oC, 5% CO2 incubator until the next day.

On the day of transfection, perform the following procedures:

7.

Inspect both of your 6-well plates, record their confluency and whether the cells
are evenly spread throughout the well
Wells should be 70-80% confluent for optimal transfection efficiency

8.

Approximately 1-3 hours prior to the addition of the transfection complexes,

remove the culture medium from all the wells of both 6-well plates and add 2 ml
of fresh (pre-warmed) Complete Medium to each well.
Note: It will take you about an hour or more to prepare your transfection mixes,
so replace the medium at the beginning of the lab period.

9.

Return plates to incubator.

10. During the incubation period, begin preparing the transfection complexes as
follows. We recommend 1 student from each pair perform the CaPO4
transfection and the other student perform the Lipofectamine 2000 transfection.

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3.1.2 CaPO4 Transfection Method
1. In sterile microfuge tubes (labeled A1-A6), aseptically mix in the following order:
pAcGFP1-C1 plasmid DNA (0.5 ug/ul), dH20, and 2.5 M CaCl2 using amounts
indicated in table below.

Tube

pAcGFP1-C1
plasmid
g

dH20 volume needed

to give final plasmid 2.5 M CaCl2
volume= 90 ul

Total
Volume

A-1

10 l

100

A-2

10 l

100

A-3

10 l

100

A-4

10

10 l

100

A-5

15

10 l

100

A-6

20

10 l

100

2.

In another set of sterile microfuge tubes, (labelled Tubes B1-B6) add 100ul of
2X HEPES Buffered Saline (HBS) to each tube.

3.

Using one P1000 set to 1000 l, slowly and continuously pipette air into Tube
B1 to create small percolating bubbles in the solution. Using a 2 nd P1000, add
the Tube A1 solution, drop by drop, to the bubbling Tube B1 solution. Continue
bubbling the mixture for an additional 15-20 seconds to create a fine CaPO4DNA precipitate. Repeat this bubbling and dropwise mixing for Tubes A2 & B2
(etc).
Ask instructor to demonstrate bubbling/mixing technique

4.

Allow the mixture to stand at room temperature for 30 minutes. A very fine
precipitate may be visible as a slight cloudiness in the solution.

5.

Vortex each tube, then add the contents of the tube dropwise and evenly over
the medium in the tissue culture wells one tube per correspondingly labeled
well.

6.

Mix gently by rocking the plate back and forth and side to side (dont swirl) and
incubate overnight in a 37oC, 5% CO2 incubator.

7.

The next morning, remove (aspirate) the medium from the wells and gently
rinse the wells 1x with PBS as follows: add 2ml of PBS, rock the plate gently
and aspirate PBS.

8.

Add 4 ml pre-warmed NIH-3T3 Complete Medium.

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9.

Incubate cultures in 37oC, 5% CO2 incubator.

10. GFP expression is highest at 24-72 hours post-transfection. Therefore,

observation and measurement of cell fluorescence, and subsequent generation
of cell lysates (see Section 3. below) should be done within the next 2 days.

3.1.3 Lipofectamine 2000 Method

1. In sterile eppendorf tubes (labeled C1-C6), gently mix pAcGFP1-C1 plasmid
DNA (0.5ug/ul) into DMEM (containing no serum) according to the table below:

Tube

pAcGFP1-C1 Plasmid
g

C1

C2

C3

C4

C5

C6

10

DMEM volume to give

final volume = 250ul

2. Prepare another set of sterile microfuge tubes (labeled D1-D6). Gently mix the
tube of Lipofectamine 2000 supplied to you, then add Lipo2000 directly into
DMEM according to the table below:

Tube

Lipofectamine 2000

D1

0 l

D2

2 l

D3

3 l

D4

10 l

D5

15 l

D6

20 l

DMEM (no FBS)

make-up volume to 250 l

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3. Incubate Lipo2000/DMEM solution for 5 minutes at room temperature.
Note: you must proceed to next step within 25 minutes
4. Combine tube C1 with D1 and mix immediately (finger vortex) before proceeding
to the next tube (repeat step 5 with Tubes C2 & D2 etc).
5. Incubate Lipo:DNA solutions for 20 minutes at room temperature to allow
complexes to form (solution may appear cloudy).
6. Pipette the contents of each tube dropwise and evenly over the medium of the
tissue culture well one tube per correspondingly labeled well.
7. Mix gently by rocking the plate back and forth and side to side (dont swirl) then
incubate plates overnight in 37oC, 5% CO2 incubator.
8. The next morning, remove (aspirate) the medium from the wells and gently rinse
each well PBS as follows: add 2ml of PBS, rock the plate gently and aspirate
PBS.
9. Add 4 ml of pre-warmed NIH-3T3 Complete Medium.
10. Incubate cultures in a 37oC, 5% CO2 incubator.
11. GFP expression is highest at 24-72 hours post-transfection. Therefore,
observation and measurement of cell fluorescence, and subsequent generation
of cell lysates (see Section D below) should be done within the next 2 days.

3.2: ANALYSIS OF TRANSIENT TRANSFECTANTS

Background to Cell labeling and Fluorescence Microscopy
Cells stained with fluorescent dyes (see Fig. 3.2) can be visualized using a
fluorescence microscope (Fig 3.1). Live dyes stain living cells and generally do not
compromise a cells viability. Some can be added to cells and taken up by them;
others, such as GFP, are expressed by the cell itself. There are literally hundreds of
dyes that can be used to stain cells once they are fixed and permeablized to allow
entry of the dye into the cell. Fixation involves the addition of organic solvents, such
as methanol; therefore, cells are killed in the process. Certain dyes can be added
directly to fixed cells to stain specific structures, such as nuclei or other organelles.
Other dyes stain specific macromolecules, such as DNA (e.g DAPI, Hoescht). Dyes
can also be used indirectly by coupling them to an antibody that recognizes a
specific protein on the cell surface (in which case fixation is not always required and
viably labelled cells may be recovered) or inside the cell (requires fixation).

If cell suspensions are labeled with a fluorescent-tagged antibody, this is referred to

as immunofluorescence. If cells are stained with dyes after being fixed or mounted
onto a microscope slide (or cell culture surface) and visualized by microscope, this
technique is called histochemistry or cytochemistry (or immunocytochemistry if an
antibody is involved in the labeling process).

Figure 3.2. Fluorescence Microscope

Figure 3.3. Excitation and Emission Spectra of Fluorescent Dyes

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Use of GFP expression (reporter) vectors to measure transfection efficiency
Transfection of a plasmid vector containing a reporter gene, such as GFP, results in
expression of the reporter (i.e. transcription and translation of the gene from the
episomal plasmid). Maximal expression (e.g. green fluorescence in the case of GFP)
occurs between 24-72 hours post-transfection, after which the expression drops off
due to degradation and/or dilution of the plasmid by continued cell division. This type
of transfection and reporter gene expression is thus referred to as a transient.
Stable transfectants can also be obtained by selecting for rare integration events of
the plasmid into the host cell genome using a selectable marker typically, an
antibiotic resistance gene, such as NeoR (e.g. present on the pAcGFP1-C1 vector).
It typically takes 2 weeks or more to obtain stable transfectants (clones), so in the
interest of time, we will be examining transient transfectants for relative GFP
expression using the fluorescence microscope and a fluorescence plate reader.

3.2.1: Analysis of Transfectants Using a Fluorescence Microscope

1. After 24 to 48 hour incubation period (post-transfection), examine transfectants
and control cells under the fluorescence microscope.
2. Switch between visible light and fluorescent light to estimate the approximate
percentage of cells in a given field that are fluorescing positive (green) for GFP.
Tabulate the % GFP+ cells for each well in your lab notebook.

3.2.2: Measurement of GFP Using a Fluorescence Plate Reader

1. After a 48-72h incubation period (post-transfection), measure the fluorescence
intensity (FI) in each 6-well using the Envision plate reader (Perkin-Elmer). FI is
the total fluorescence being emitted by all cells in a given well and has arbitrary
units. The settings of the plate reader are adjusted to attain the best signal to
noise ratio (of each transfection well vs the control well). Instructor will
demonstrate the instrument.
Instrument Measurement Specs:
Excitation & emission light is generated and measured, respectively, using a
bottom mirror (i.e. light shines up through the bottom of the plate, where the
cells are attached). Record the filter set & instrument specs used for your plate
measurement:
Excitation filter: ___________________________ nm
Emission filter: ___________________________ nm
Gain: ________________
Fluorescence Intensity (FI): ____________ %

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2. Record fluorescence intensity (FI) counts in your lab notebook. Subtract
background FI of the control well from the FI of the test wells to obtain an
estimate of relative FI for each sample.

3.2.3: Preparation of Cellular Lysates (for Western Blot Analysis)

Materials and Reagents

PBS (ice cold; doesnt need to be sterile)
Lysis Buffer (each pair requires 5 ml):
50 mM Hepes pH 7.2
50 mM NaCl
5 mM EDTA
1 % Triton

(protease inhibitors, added fresh)

20 ug/mL Leupeptin
20 ug/mL Soy bean trypsin inhibitor
20 ug/mL Pepstatin
80 ug/mL PMSF

Procedure
Note: Perform steps 1 & 2 in a biological hood. The rest of the procedure can be
done outside the hood, since cells are no longer alive:
1. After obtaining a complete set of live cell fluorescence data from your
transfection plates (i.e. from microscope & plate reader), remove the medium in
the wells and gently rinse cells with 2 ml of PBS (avoid aspirating cells that may
have lifted off the plate by placing pipette in a different area of the well).
2. Using a Gilson pipette and clean tip, add 300ul of ice cold lysis buffer to each
well (protease inhibitors must be freshly added to lysis buffer just prior to use)
3. Place plates on ice for 5 minutes make sure plate is sitting level so that cells
are evenly covered with buffer.
4. Gently pipette lysis solution up and down 6X, then transfer lysate to a clean
microfuge tube.
5. Centrifuge tube in microfuge for 5 minutes at top speed (13,000 rpm) to pellet
insoluble debris.
6. Transfer supernatant to new tube, place in designated storage box that will be
kept at -20oC until analysis (several weeks from now).

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3.2.4: Reducing SDS-PAGE of Cellular Lysates
Unpolymerized acrylamide is a toxic substance, so gloves must be worn while
handling gels or solutions that contain it. Report any spills containing acrylamide.

Materials and Reagents

One Pre-cast (commercial) SDS polyacrylamide gel (take note of % in separating

gel)

12 Samples = cell lysates prepared from transfected cells and stored frozen (see
Section 3.2.3)

For 2 groups of students or 2 gels, prepare 500 mL of 1X Gel Running Buffer

from the 10X stock provided

Procedure
Gel prep:
1. Each gel tank holds 2 gels and requires 500 mL of Running (or Electrode) Buffer,
so coordinate with another group and prepare 1X buffer in a graduated cylinder.
Use a magnetic stirrer to mix in the grad cylinder while you prepare the gel.
2. Insert pre-cast gel into the BioRad Minigel apparatus as demonstrated. Each
group will need to set-up one gel (two groups will join into a single gel tank for
running 2 gels).
3. Use a transfer pipette to gently fill and rinse gel sample wells with 1X Running
Buffer. It is very important to remove unpolymerized acrylamide; otherwise wells
will be clogged and you wont be able to load your samples properly. Invert gel
to remove rinse and use a kimwipe to wick away any remaining liquid (wells
should be empty).
4. Join with another group to assemble 2 gels into a gel tank and form the
electrophoresis chamber. If you dont have a 2nd gel, use a dummy plate to form
the buffer chamber.
5. Place the gel assembly into the buffer tank and pour 1X running buffer into the
tank until the buffer level is ~1 inch above the bottom of the plates, then fill the
inside chamber (formed by the 2 gels) with 1X running buffer. Check to ensure
that the top buffer is not leaking (buffer level should not drop significantly.

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Sample Prep & Gel Loading:
6. Thaw cell lysates (generated from your GFP-transfectants in Section 3.2.3) and
pipette 24 uL of each lysate into a labelled microfuge tube.
7. Add a 1/5 volume of 5X SDS-PAGE gel loading buffer (containg
mercaptoethanol as reducing agent) to achieve a final sample volume of 30uL.
8. Heat samples for 5 min in a 95C or boiling water bath to completely denature
the proteins.
9. Cool samples at room temperature for 2 minutes, then pulse spin in a microfuge
to collect any evaporated liquid.
10. Use a P20 to load samples into the gel wells, recording the sample key in your
notebook.
11. Use an outside well to load 1 lane of pre-stained molecular weight standards
(volume & specifications will be provided by instructor).
12. Plug apparatus into the power supply and run at 40mAmps per gel constant
current (for 2 gels, use 80mAmps constant current).

13. Run samples until the dye front reaches the bottom of the separating gel.
14. While gel is running, prepare Transfer Buffer and containers for Western Blotting
(see next section).
15. Also prepare 3mm Whatman and nitrocellulose (NC) blotting paper as follows:
a. Use clean scissors and wear gloves when you handle the paper & NC
b. Keep NC between the wax paper to protect it; this is the blot onto which
proteins from your gel will be transferred
c. Use a gel dummy plate, ruler & pencil to measure & cut 8 pieces of 3mm
Whatman paper and 1 piece of nitrocellulose (NC), each matched to the size
of the gel.

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3.2.5: Western Blotting of SDS-PAGE Gel (Elecrophoretic Transfer of Proteins)

Materials and Reagents

Transfer Buffer (TB):
Tris base
5.81 g
Glycine
2.93 g
SDS
0.375 g
Methanol
200 ml
Make-up to 1000 mL with distilled H20 and stir until dissolved
Nitrocellulose paper (blot) 1 piece cut to same size as gel
Whatman filter paper 8 pieces cut to same size as gel
2 clean Tupperware containers for soaking gel or paper
Western Blotting Apparatus including frozen cooling block
Power pack

Procedure
1. Following electrophoresis, disassemble gel apparatus and use the flat edge of a
clean spatula to carefully pry apart gel plates (gel will stick to one side).
2. Use the flat edge of a clean spatula to lift a corner of the gel and peel it into a
container containing transfer buffer (TB). Allow gel to equilibrate for a few
minutes.
3. Half fill a 2nd tupperware container with TB buffer and use this for wetting the
Scotchbrite pads (of the Western cassette), Whatman & NC paper.
4. From your 8 (pre-cut) pieces of Whatman paper, form two even stacks of 4
pieces each.
5. Dip 1 Scotchbrite pad into TB to completely wet it and place it on the grey/black
side of the cassette. Dip 1 stack (of 4) Whatman into TB to completely wet the
paper and place stack ontop of the Scotchbrite pad.
6. Pre-wet your gloved fingers in the TB containing your gel, then slide both sets of
fingers underneath the gel to carefully lift it out of the container and position it
ontop of the wetted stack of Whatman on the cassette. Dont tug at the gel to
move it instead, keep the gel wet and slide your wetted fingers underneath the
gel to lift and re-position it.

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7.

Use clean, flat-edged (Millipore) forceps to transfer the NC paper into TB.
Completely wet the NC paper there should be no white spots, which indicate
contaminants (fingerprints) on the paper. Place NC ontop of the wetted gel on
the cassette.

8.

Dip 2nd stack (of 4) Whatman into TB to completely wet the paper and place
stack ontop of the NC paper. Use a clean test tube or pipette like a rolling pin
along the surface of the stack to smooth away any bubbles that may be
trapped between the gel and NC paper (which will prevent transfer of proteins).

9.

The resultant stack in the blotting cassette should have a polarity of gel NC
going from negative to positive (i.e. gel nearest to grey/black side of cassette,
NC nearest to clear/red side of cassette). Once you have confirmed correct
polarity, close the cassette using the sliding locks.

10. Fill the blotting chamber about halfway with TB, then add the ice pack and
blotting cassette(s). Top-up chamber with TB if necessary.
11. Plug apparatus into powerpack and transfer proteins at 0.8 mA x area (cm2) of
the gel (14pprox.. 40 mA for a mini gel) for 1 hour.
12. Disassemble blotting sandwich and peel NC paper (= protein blot) away from gel
using a clean pair of flat-edged (Millipore) forceps.
13. Place NC blot on a clean piece of Whatmann and allow to air dry
14. Trace over the pre-stained molecular weight standards on the blot with a soft
pencil.
15. Sandwich blot between 2 fresh sheets of (dry) 3mm Whatman paper and wrap
in plastic wrap. Store at RT until next lab.

3.2.6: Western Blot Analysis (Immunoblotting) for Detection of GFP Protein

Materials and Reagents
Ponceau S protein stain (optional)
Tris buffered Saline (TBS):
25 mM Tris-HCl
3g
NaCl
8g
M KCl
0.2 g
Adjust pH to 7.4
Make-up volume to 1L in distilled H20

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Blocking buffer: 5% BSA (molecular biology grade) in TBS

Wash buffer: 0.1 % Tween-20 in TBS
Primary antibody solution: Rabbit anti-GFP antibody diluted 1/1000 in Wash buffer +
1% BSA just prior to use.
Secondary Antibody Solution: Goat anti-rabbit IgG antibody conjugated to horseradish peroxidase (HRP) diluted 1/5000 to 1/10,000 in Wash buffer + 1% BSA just
prior to use.

Procedure
1. Optional: Stain blot with Ponceau S (~ 3 minutes), then destain briefly with water.
Take a picture or photocopy the blot as a record try not to let the blot dry out.
2. Place blot in a Seal-a-meal bag containing 5 mL of Blocking buffer and incubate
at RT with rocking for a minimum of 1 hr or O/N (BSA in buffer will prevent nonspecific antibody:protein interactions).
3. Remove blot from blocking solution and put into a new Seal-a-meal bag
containing 4ml of Primary Antibody solution (containing freshly diluted anti-GFP
antibody) and incubate at RT with rocking for a minimum of 1 hr or O/N.
4. Wash blot 3x with Wash buffer: 50 mL, 10 min per wash.
5. Place blot in a new Seal-a-meal bag containing 4ml of Secondary Antibody
solution (containing freshly added HRP-conjugated Ab) and incubate for1 hour at
RT.
6. Wash blot 3x in Wash buffer: 50mL, 10 min per wash
7. Wash blot 1x in 50 mL of TBS for 5 min (to remove Tween).
8. Perform enhanced chemiluminescent detection (ECL) according to
manufacturers protocol (see Amersham manual; handed out in lab).

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3.3: ASSIGNMENT: LAB 3 DATA SUMMARY

3.3.1

Due Date

Data Summary is due 2 weeks after the completion of Lab 3 (usually the 3rd week in
March)

Given the big gap between Sections 3.2 & 3.3 of this lab (i.e. fluorescence
measurements and Western blot), it is highly recommended that you analyze &
collate your data (e.g. prepare summary Table) as you acquire them. Its also a
good idea to write-up the experimental approach for each section, as you go, so
you remember what you did

There are often Midterms happening just before & after Spring break, so get a
headstart by completing all but the Western Blot portion of your report

3.3.2

Data gathering

Each student must prepare an independent Data Summary, so you will need a
scanned image (or original photograph) of the Western Blot, along with all the
fluorescence microscope and plate reader data.

Obtain any data recorded in your partners notebook the same day they are
gathered, so you wont be missing data when the time comes to complete your
lab report.

Heed this advice so you wont be hunting for data at midnight before your
summary is dueit happens every year!

3.3.2

Data Summary Template & Guidelines

If you adhere to the guidelines below, your Data Summary should be between 4 to
4.5 pages. I am setting a 5 page maximum. If you are having trouble staying within
5 pages, please come talk to me and I will help you achieve the appropriate level of
relevant detail.
General Guidelines

The purpose of a Data Summary is to give the reader (typically a supervisor) a

concise snapshot of the experiment you performed, the results and your final
conclusion about whether the stated purpose was achieved (or not)
Rule #1: imagine yourself as the reader who is familiar with cell biology but
had no involvement with your experiment and hasnt yet read the protocol. If
you do a good job they wont need to read the protocol or refer to your
notebook (trust me, it is best to avoid a boss looking through your notebook!)
you need to walk the reader through your approach, what you did
experimentally (in chronological order), the results and interpretation

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however, the data summary should not be a repeat of your notebook it
should contain summary data only and an overview of the experimental
approach (see template for specific guidelines under each section).

Organization, grammar and spelling count not just to me but to future bosses.
Written reports are used to judge your competency and level of understanding,
so even if you DO understand the experiment but cant communicate what you
did in a clear and concise manner, supervisors will assume youre confused or
dont get it.
because a Data Summary describes experiments that are completed, use
past tense and a passive voice: The purpose of this experiment was to.,
Samples were loaded into. Avoid inserting yourself into the report, We
loaded samples.
If you label figures completely and use legends for procedural details, you
dont need to write as much in the body of the report and this is a great trick
for those of you who struggle with English grammar

Template to be Used for Lab 3 Data Summary

Below is the template to be followed for preparing a concise Data Summary. See guidelines
& examples in each section for details about what should be included. Equally important is
what should not be included; please adhere to the section guidelines.

Use the following 5 sections (only) and try to stay within the page recommendations

Use 1.5 line spacing and a minimum of font 10 for the main text

Figure legend(s), supplementary text under Table(s) and labels should be in a smaller
font. Section titles should be bold-typed, capitalized or underlined etc.

PURPOSE

A few sentences stating what you were trying to achieve and/or test in the experiment

EXPERIMENTAL APPROACH

1-1.5 pages

This is NOT a full Materials & Methods section. It is a chronological overview of the
entire experiment and the methods used.

You may refer to the Lab 3 protocol for complete details of general procedures.
However, include particulars of your experiment that are important for understanding the
experiment, interpreting the results. Also include specifics that the reader could not
know by reading the lab protocol (e.g. the passage #, VCD and % viability of your 3T3
seed culture, # of cells seeded for transfection etc).

For buffers or reagents that need to be mentioned, state their final composition OR put
these details in the figure legend(s) (e.g. % of the SDS gel, final dilutions and type of
antibodies used etc can go in the Fig legend beneath the blot).

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RESULTS
2 to 2.5 pages

Do NOT include raw data or transcribe working tables from the lab protocol, which
contain all the small details of the experiment. Your supervisor isnt interested in how
much water or DMEM was used to make-up a final volume. Instead, state the final
volume or concentration of common components (in the Experimental Approach or
figure legend) and present the relevant variable being tested (compared) in your data
tables or figures.

Since the same set of conditions was compared using more than one readout/analysis, the
data can be consolidated into a summary Table(s) including: final amounts or
concentrations of the relevant variable(s) in one column and the associated calculated
(not raw) values from each type of analysis in adjacent columns.

Note: if you want to split Lipo & CaCl2 transfection data into 2 Tables that is okay too.
Briefly describe what is presented in Table 1.
Table 1. Title

Include any specifics about measurements or raw data manipulations beneath the Table in smaller font. Include
a sample calculation for the values presented (if needed).

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Give a concise explanation of what the data in Table 1 show:
e.g. The data presented in Table 1 indicate that samples x and y contained the greatest
number of the GFP+ cells...
Save broader interpretations for the totality of your data for the Discussion section, where
you can also discuss any experimental errors that may explain odd or inconsistent data.

Insert a scanned image (preferred) or paste in a photo of your Western blot. Number lanes of
gel and include a sample key beside gel (as shown) or in Figure legend. Place tick marks
next to each MW standard and its size (in kDa).

Describe what is presented in Figure 1

Figure 1. Title
MW (kDa)

10

11

12

Lane

Sample

Figure 1 (legend is written in smaller font, placed beneath figure): Add any details about your
samples, gel, blot, detection method etc that help the reader understand the particulars of the analysis
you performed (there is more than 1 way to do a Western blot). Be sure to include specifics that are
not included in the lab protocol. Note, these are the details not generally included (i.e. repeated) in the
Experimental Approach, b/c they are more relevant next to the figure.

Briefly state what your results from the Western Blot (in Figure 1) show.

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DISCUSSION
1 to 1.5 pages

Interpret your results and discuss whether the data from each analysis agree with one
another (i.e. do the data show the same trend?)

With your purpose in mind, reason through a conclusion based on the totality of your
data OR make a conclusion based on only a subset of your data. If you decide to weigh
some data more in making a conclusion, this is okay but state your case (i.e. why you
trust these data more).

For unexpected results or less than ideal data, suggest possible reasons. This is the
section where you can troubleshoot and discuss aspects of the experiment or procedure
you think should be modified and/or repeated and why. You are encouraged to suggest
ways to optimize the procedures and/or alternative approaches to achieve the stated
purpose.

Do not discuss vague human or experimental error. They teach you this in high
school but everyone in science knows about these inherent errors, so were done talking
about them! If you did make a mistake in a particular procedure that impacted the data,
briefly explain that an incorrect amount was used and how this specifically affected a
transfection condition or lane in the gel etc.