Lab 3_Transfection NIH-3T3 (2013)

8/10/2019 Lab 3_Transfection NIH-3T3 (2013)

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Lab 3

Optimization of Transfection UsingCaPO4& Lipofectamine 2000 Methods

Animal Cell BiologyBIOT 4230

Winter, 2013


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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 (typicallyplasmid vectors) into cells is termed transfection. The most common transfectionmethods involve the formation of chemical complexes of DNA with a variety ofreagents, 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 todetermine 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 optimizetransfection 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 H2OSterile microfuge tubes

Set of Gilson pipettes & sterile tips

CaPO4Method:

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 d ay before transfect ion, seed tw o 6-well plates as fol lows:

1. Inspect the NIH-3T3 flasks you have been maintaining over the past 2 weeksand select 2 or 3 of your most confluent T25 flasks that have been in continuouslogarithmic growth

Guidelines: you will need 5 x 106total cells for seeding and one 95% confluentT25 flask contains ~2.5 to 3 x 106total cells

2. Trypsininze and block all of your chosen flasks to generate a single-cellsuspension (SCS) as follows:

a. Follow the trypsinization procedure described in Lab 2, except pool yourSCS (from each flask) into a sterile conical test tube

b. Keep the final volume of your pooled single cell suspension between 20 to24 mL

c. If you are harvesting from 2 flasks, block with 8 mL of Complete Medium andpool 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 andpool 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 (asper Supplementary protocol, Cell density and viability assessment) anddetermine 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 4x10

5 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 timesto resuspend the cells, then use a 10 mL pipette to seed 4x105 cells into all wellsof 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 1plate for the CaPO4transfection

6. Incubate plates in 37oC, 5% CO2incubator until the next day.

On the day of transfect ion, perform the fol lowing p rocedures:

7. Inspect both of your 6-well plates, record their confluency and whether the cellsare 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 mlof 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 asfollows. We recommend 1 student from each pair perform the CaPO4transfection and the other student perform the Lipofectamine 2000 transfection.


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3.1.2 CaPO4Transfection 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 amountsindicated in table below.

Tube

pAcGFP1-C1plasmid

dH20 volume neededto give final plasmid

volume= 90 ul2.5 M CaCl2

TotalVolume

g l

A-1 0 10 l 100

A-2 1 10 l 100

A-3 5 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 of2X HEPES Buffered Saline (HBS) to each tube.

3. Using one P1000 set to 1000 l, slowly and continuously pipette air into TubeB1 to create small percolating bubbles in the solution. Using a 2ndP1000, addthe Tube A1 solution, drop by drop, to the bubbling Tube B1 solution. Continuebubbling the mixture for an additional 15-20 seconds to create a fine CaPO4-

DNA 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 fineprecipitate may be visible as a slight cloudiness in the solution.

5. Vortex each tube, then add the contents of the tube dropwise and evenly overthe medium in the tissue culture wells one tube per correspondingly labeledwell.

6. Mix gently by rocking the plate back and forth and side to side (dont swirl)andincubate overnight in a 37oC, 5% CO2incubator.

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

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


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9. Incubate cultures in 37oC, 5% CO2incubator.

10. GFP expression is highest at 24-72 hours post-transfection. Therefore,observation and measurement of cell fluorescence, and subsequent generationof 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 plasmidDNA (0.5ug/ul) into DMEM (containing no serum) according to the table below:

TubepAcGFP1-C1 Plasmid

DMEM volume to givefinal volume = 250ul

g lC1 0

C2 1

C3 1

C4 5

C5 5

C6 10

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

Tube Lipofectamine 2000DMEM (no FBS)

make-up volume to 250 l

D1 0 l

D2 2 l

D3 3 l

D4 10 l

D5 15 l

D6 20 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 proceedingto the next tube (repeat step 5 with Tubes C2 & D2 etc).

5. Incubate Lipo:DNA solutions for 20 minutes at room temperature to allowcomplexes to form (solution may appear cloudy).

6. Pipette the contents of each tube dropwise and evenly over the medium of thetissue culture wellone tube per correspondingly labeled well.

7. Mix gently by rocking the plate back and forth and side to side (dont swirl) thenincubate plates overnight in 37

oC, 5% CO2incubator.

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 aspiratePBS.

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

10. Incubate cultures in a 37oC, 5% CO2incubator.

11. GFP expression is highest at 24-72 hours post-transfection. Therefore,observation and measurement of cell fluorescence, and subsequent generationof cell lysates (see Section D below) should be done within the next 2 days.

3.2: ANALYSIS OF TRANSIENT TRANSFECTANTS

Backgro und to Cel l labeling and Fluorescence Microscopy

Cells stained with fluorescent dyes (see Fig. 3.2) can be visualized using afluorescence microscope (Fig 3.1). Live dyesstain living cells and generally do notcompromise 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 ofdyes that can be used to stain cells once they are fixed and permeablized to allowentry of the dye into the cell. Fixation involves the addition of organic solvents, suchas methanol; therefore, cells are killed in the process. Certain dyes can be addeddirectlyto fixed cells to stain specific structures, such as nuclei or other organelles.Other dyes stain specific macromolecules, such as DNA (e.g DAPI, Hoescht). Dyescan also be used indirectly by coupling them to an antibody that recognizes aspecific protein on the cell surface (in which case fixation is not always required andviably labelled cells may be recovered) or inside the cell (requires fixation).


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If cell suspensions are labeled with a fluorescent-tagged antibody, this is referred toas immunofluorescence. If cells are stained with dyes after being fixed or mountedonto a microscope slide (or cell culture surface) and visualized by microscope, thistechnique 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 m easure transfect ion eff ic iency

Transfection of a plasmid vector containing a reporter gene, such as GFP, results inexpression of the reporter (i.e. transcription and translation of the gene from theepisomal plasmid). Maximal expression (e.g. green fluorescence in the case of GFP)

occurs between 24-72 hours post-transfection, after which the expression drops offdue to degradation and/or dilution of the plasmid by continued cell division. This typeof transfection and reporter gene expression is thus referred to as a transient.Stable transfectants can also be obtained by selecting for rare integration events ofthe plasmid into the host cell genome using a selectable marker typically, anantibiotic 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 theinterest of time, we will be examining transient transfectants for relative GFPexpression 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 transfectantsand control cells under the fluorescence microscope.

2. Switch between visible light and fluorescent light to estimate the approximatepercentage 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 fluorescenceintensity (FI) in each 6-well using the Envision plate reader (Perkin-Elmer). FI isthe total fluorescence being emitted by all cells in a given well and has arbitraryunits. The settings of the plate reader are adjusted to attain the best signal tonoise ratio (of each transfection well vs the control well). Instructor willdemonstrate the instrument.

Instrum ent Measurement Specs:

Excitation & emission light is generated and measured, respectively, using abottom 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 platemeasurement:

Excitation filter: ___________________________ nm

Emission filter: ___________________________ nm

Gain: ________________

Fluorescence Intensity (FI): ____________ %


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

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


PBS (ice cold; doesnt need to be sterile)

Lysis Buffer (each pair requires 5 ml):(protease inhibitors, added fresh)

50 mM Hepes pH 7.2 20 ug/mL Leupeptin

50 mM NaCl 20 ug/mL Soy bean trypsin inhibitor5 mM EDTA 20 ug/mL Pepstatin1 % Triton 80ug/mL PMSF

Procedure

Note: Perform steps 1 & 2 in a biological hood. The rest of the procedure can bedone outside the hood, since cells are no longer alive:

1. After obtaining a complete set of live cell fluorescence data from yourtransfection plates (i.e. from microscope & plate reader), remove the medium inthe wells and gentlyrinse cells with 2 ml of PBS (avoid aspirating cells that mayhave 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 eachwell (protease inhibitors must be freshly addedto lysis buffer just prior to use)

3. Place plates on ice for 5 minutes make sure plate is sitting level so that cellsare evenly covered with buffer.

4. Gently pipette lysis solution up and down 6X, then transfer lysate to a cleanmicrofuge tube.

5. Centrifuge tube in microfuge for 5 minutes at top speed (13,000 rpm) to pelletinsoluble debris.

6. Transfer supernatant to new tube, place in designated storage box that will bekept 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 whilehandling gels or solutions that contain it. Report any spills containing acrylamide.


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

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

For 2 groups of students or 2 gels, prepare500 mL of 1X Gel Running Bufferfrom 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. Eachgroup will need to set-up one gel (two groups will join into a single gel tank forrunning 2 gels).

3. Use a transfer pipette to gently fill and rinse gel sample wells with 1X RunningBuffer. It is very important to remove unpolymerized acrylamide; otherwise wellswill be clogged and you wont be able to load your samples properly. Invert gelto remove rinse and use a kimwipe to wick away any remaining liquid (wellsshould be empty).

4. Join with another group to assemble 2 gels into a gel tank and form theelectrophoresis chamber. If you dont have a 2

ndgel, use a dummy plate to form

the buffer chamber.

5. Place the gel assembly into the buffer tank and pour 1X running buffer into thetank until the buffer level is ~1 inch above the bottom of the plates, then fill theinside chamber (formed by the 2 gels) with 1X running buffer. Check to ensurethat 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) andpipette 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 denaturethe proteins.

9. Cool samples at room temperature for 2 minutes, then pulse spin in a microfugeto collect any evaporated liquid.

10. Use a P20 to load samples into the gel wells, recording the sample key in yournotebook.

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 constantcurrent (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 whichproteins from your gel will be transferred

c. Use a gel dummy plate, ruler & pencil to measure & cut 8 pieces of 3mmWhatman paper and 1 piece of nitrocellulose (NC), each matched to the sizeof the gel.


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


Transfer Buffer (TB):Tris base 5.81 gGlycine 2.93 gSDS 0.375 gMethanol 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 paper8 pieces cut to same size as gel

2 clean Tupperware containers for soaking gel or paperWestern Blotting Apparatus including frozen cooling block

Power pack

Procedure

1. Following electrophoresis, disassemble gel apparatus and use the flat edge of aclean 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 peelit into acontainer containing transfer buffer (TB). Allow gel to equilibrate for a fewminutes.

3. Half fill a 2nd tupperware container with TB buffer and use this for wetting theScotchbrite pads (of the Western cassette), Whatman & NC paper.

4. From your 8 (pre-cut) pieces of Whatman paper, form two even stacks of 4pieces each.

5. Dip 1 Scotchbrite pad into TB to completely wet it and place it on the grey/blackside of the cassette. Dip 1 stack (of 4) Whatman into TB to completely wet thepaper and place stack ontop of the Scotchbrite pad.

6. Pre-wet your gloved fingers in the TB containing your gel, then slide both sets offingers underneath the gel to carefully lift it out of the container and position itontop of the wetted stack of Whatman on the cassette. Dont tug at the gel tomove itinstead, keep the gel wet and slide your wetted fingers underneath thegel 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 paperthere should be no white spots, which indicatecontaminants (fingerprints) on the paper. Place NC ontop of the wetted gel onthe cassette.

8. Dip 2

nd

stack (of 4) Whatman into TB to completely wet the paper and placestack ontop of the NC paper. Use a clean test tube or pipette like a rolling pinalong the surface of the stack to smooth away any bubbles that may betrapped 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 NCgoing 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 correctpolarity, 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) ofthe gel (14pprox.. 40 mA for a mini gel) for 1 hour.

12. Disassemble blotting sandwich and peel NC paper (= protein blot) away from gelusing 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 softpencil.

15. Sandwich blot between 2 fresh sheets of (dry) 3mm Whatman paper and wrapin plastic wrap. Store at RT until next lab.

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


Ponceau S protein stain (optional)

Tris buffered Saline (TBS):

25 mM Tris-HCl 3 g

NaCl 8 g

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 horse-radish peroxidase (HRP) diluted 1/5000 to 1/10,000 in Wash buffer + 1% BSA justprior 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 recordtry not to let the blot dry out.

2. Place blot in a Seal-a-meal bag containing 5 mL of Blocking buffer and incubateat RT with rocking for a minimum of 1 hr or O/N (BSA in buffer will prevent non-specific antibody:protein interactions).

3. Remove blot from blocking solution and put into a new Seal-a-meal bagcontaining 4ml of Primary Antibody solution (containing freshly diluted anti-GFPantibody) and incubate at RT with rocking for a minimum of 1 hr or O/N.

4. Wash blot 3xwith Wash buffer: 50 mL, 10 min per wash.

5. Place blot in a new Seal-a-meal bag containing 4ml of Secondary Antibodysolution (containing freshly added HRP-conjugated Ab) and incubate for1 hour atRT.

6. Wash blot 3xin 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 tomanufacturers protocol (seeAmershammanual; 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. I ts also agood idea to write-up the experimental approach for each section, as you go, soyou remember what you did

There are often Midterms happening just before & after Spring break, so get aheadstart 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 ascanned image (or original photograph) of the Western Blot, along with all thefluorescence microscope and plate reader data.

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

Heed this advice so you wont be hunting for data at midnight before yoursummary 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 to4.5 pages. I am setting a 5 page maximum. If you are having trouble staying within5 pages, please come talk to me and I will help you achieve the appropriate level ofrelevantdetail.

General Guidel ines

The purpose of a Data Summary is to give the reader (typically a supervisor) aconc ise 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 buthad no involvement with your experiment and hasnt yet read the protocol. Ifyou do a good job they wont need to read the protocol or refer to yournotebook (trust me, it is best to avoid a boss looking through your notebook!)

you need to walk the reader through your approach, what you didexperimentally (in chronological order), the results and interpretation


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however, the data summary should not be a repeat of your notebook itshould contain summary data only and an overview of the experimentalapproach (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 youdid in a clear and concise manner, supervisors will assume youre confused ordont get it.

because a Data Summary describes experiments that are completed, usepast tense and a passive voice: The purpose of this experiment was to.,Samples were loaded into. Avoid inserting yourself into the report, Weloaded samples.

If you label figures completely and use legends for procedural details, youdont 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


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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 iswhat 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 smallerfont. Section titles should be bold-typed, capitalized or underlined etc.

PURPOSE

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

EXPERIMENTAL APPROACH

1-1.5 pages

This is NOT a full Materials & Methodssection. 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

seedculture, # 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 howmuch 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 & CaCl2transfection 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 greatestnumber of the GFP

+cells...

Savebroader interpretations for the totality of your data for the Discussionsection, 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) 1 2 3 4 5 6 7 8 9 10 11 12 Lane Sample

Figure 1 (legend is written in smaller font, placed beneath figure): Add any details about yoursamples, 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

notincluded 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.

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