EDVO-Kit # Exploring Biotechnology with the Green Fluorescent … · Biotechnology with the Green...

EVT 2012_03_09AM

EDVO-Kit #

303Exploring Biotechnology with the Green Fluorescent Protein

Storage: See Page 3 for specifi c storage instructions

EXPERIMENT OBJECTIVE:

The objective of this set of experiments is to develop an understanding of bacterial transformation by pGFP plasmid DNA, and purifi cation and characterization

of the recombinant protein.

The Biotechnology Education Company ®

EDVOTEK, Inc. • 1-800-EDVOTEK • www.edvotek.com

Updated

Revised

and

Exploring Biotechnology with the Green Fluorescent Protein2

xxx303EDVO-Kit #

EDVOTEK - The Biotechnology Education Company® • 1-800-EDVOTEK • www.edvotek.com

Duplication of any part of this document is permitted for non-profi t educational purposes only. Copyright © 2006-2012 EDVOTEK, Inc., all rights reserved. EVT 2012_03_09AM

Table of Contents

EDVOTEK, The Biotechnology Education Company, and InstaStain are registered trademarks of EDVOTEK, Inc.

Page

Experiment Components 3

Experiment Requirements 3

Background Information 5

Experiment Procedures Experiment Overview and General Instructions 10 Module I: Transformation of E. coli with pGFP 12 Module II: Isolation of GFP 16 Module III: Purifi cation of GFP by Column Chromatography 18 Module IV: Analysis of GFP by Denaturing SDS-Gel Electrophoresis 20 Study Questions 28

Instructor's Guidelines

Notes to the Instructor 29

Pre-Lab Preparations 32

Experiment Results and Analysis 40

Study Questions and Answers 42

Material Safety Data Sheets 44

Exploring Biotechnology with the Green Fluorescent Protein 3

EDVOTEK - The Biotechnology Education Company® 1.800.EDVOTEK • www.edvotek.com

FAX: 202.370.1501 • email: [email protected]

303EDVO-Kit #


Storage

Components for Transformation

A Fluorocells™ (DO NOT FREEZE) Refrigerator• Cell Reconstitution Media Refrigerator B Supercoiled pGFP Freezer C Ampicillin Freezer D IPTG FreezerE CaCl2 Room temp.

• ReadyPour™ Luria Broth Agar, sterile (2 bottles) (also referred to as ReadyPour medium) Room temp.• Luria Broth Medium for Recovery, sterile (also referred to as Luria Recovery Broth) Room temp.• Petri plates, small• Petri plates, large • Plastic microtipped transfer pipets• Wrapped 10 ml pipet (sterile)• Wrapped 1 ml pipets (sterile)• Toothpicks (sterile)• Inoculating loops (sterile)• Microcentrifuge tubes• Microtiter plate

Components for Purifi cation

F Cell Extract containing GFP (control) FreezerG TEG buffer (Tris, EDTA, Glucose) FreezerH Column Elution Buffer (10x) FreezerI Protein Molecular Weight Standards FreezerJ Dry Molecular Sieve Matrix Room temp.K Protein Denaturing Solution FreezerL 50% Glycerol Solution Freezer• Tris Glycine SDS Electrophoresis Buffer Room temp.• Protein InstaStain® Room temp.• Chromatography Columns Room temp.

Experiment Components

Component Quantities:

This experiment is designed for 6 groups.

Polyacrylamide gels are not included for the electropho-resis part of this experiment. The experiment is designed for two student groups to share a gel. A total of 3 poly-acrylamide gels (Precast poly-acrylamide gels, Cat. #651) are required.

All components are intended for educational research only. They are not to be used for diagnostic or drug purposes, nor administered to or consumed by humans or animals.

THIS EXPERIMENT DOES NOT CONTAIN HUMAN DNA. None of the experiment components are derived from human sources.

Important READ ME!

Transformation experiments contain antibiotics which are used for the selection of transformed bacteria. Students who have allergies to antibiotics such as penicillin, ampicillin, kanamycin or tetracycine should participate in this experiment at their own discretion.

Exploring Biotechnology with the Green Fluorescent Protein4

xxx303EDVO-Kit #

EDVOTEK - The Biotechnology Education Company® • 1-800-EDVOTEK • www.edvotek.com


Online Orderingnow available

Visit our web site for information about EDVOTEK’s complete line of “hands-on” experiments forbiotechnology and biology education.

Mon - Fri 9 am - 6 pm ET

(1-800-338-6835)

EDVO-TECH SERVICE

1-800-EDVOTEK

Mon - Fri9:00 am to 6:00 pm ET

FAX: 202-370-1501Web: www.edvotek.comemail: [email protected]

Please have the following information ready:

• Experiment number and title• Kit lot number on box or tube• Literature version number (in lower right corner)• Approximate purchase date

Technical ServiceDepartment

Requirements

Requirements for Transformation

• Automatic Micropipet (5-50 µl) and tips• Two Water baths (37° C and 42° C)• Thermometer • Incubation Oven (37° C)• Pipet pumps or bulbs• Ice• Marking pens• Bunsen burner, hot plate or microwave oven• Hot gloves• Long wave U.V. light (EDVOTEK cat #969 recommended)

Requirements for Purifi cation

• Vertical Gel Electrophoresis Apparatus (MV10 or MV20)• D.C. Power Source• Automatic Micropipet (5 -50 µl) and Tips (Cat. #638 Fine Tip Micropipet Tips recommended)• Balance• Ice Buckets and Ice• Long U.V. lamps (Cat. #969)• Ring stands and column clamps• 1 ml pipets and pipet pumps• Microtest tubes• Polyacrylamide gels (3)*• Glacial acetic acid• Methanol• Distilled water

* Polyacrylamide gels are not included for the elec-trophoresis part of this ex-periment. The experiment is designed for two student groups to share a gel. A to-tal of 3 polyacrylamide gels (Precast polyacrylamide gels, Cat. #651) are required.


303EDVO-Kit #

The Biotechnology Education Company® • 1-800-EDVOTEK • www.edvotek.com


Backg

rou

nd

Info

rmatio

n

The Green Fluorescent Protein (GFP)

The jelly fi sh Aquorea victoria is the natural host of the green fl uorescent protein (GFP). A bright burst of light is observed when energy is transferred to the green fl uorescent protein (GFP) located in specialized photogenic cells in the base of the jellyfi sh umbrella. This family of proteins has been known for some time and signifi cant research in this area has been reported. Fluo-rescent proteins can be expressed both in prokaryotic and eucaryotic cells. These proteins do not require substrates, other gene products, or cofactors. When exposed to long wave U.V. light, they emit a bright green light that is visible in bacteria transformed by plasmids that contain the genes encoding GFP. Likewise, purifi cation of the GFP from bacterial cell extracts is simplifi ed by their fl uorescence.

In cell biology experiments, GFP is often fused to proteins to study various biochemical processes. There are many examples of such chimeric fusion proteins using the GFP protein as a biological tag. Such fusions are either at the amino or carboxyl N- or C- termini. The chimeric proteins are used as biotechnological tools to study protein localization and traffi cking within cells.

The green fl uorescent protein (GFP) has a molecular weight of approxi-mately 40,000 daltons. Most of the intact protein is required for maintaining fl uorescence. The chromophore responsible for light emission is within the structure of the GFP protein and resides in amino acid residues 65 to 67, a cyclic tripeptide composed of Ser-Tyr-Gly. The importance of protein folding is clearly demonstrated in that GFP is fl uorescent only upon proper confor-mational folding.

With the 3-D structure of GFP being determined, several other variants of the GFP have been constructed using site-directed mutagenesis (SDM). SDM allows specifi c (point) mutations to be introduced in a protein to determine the impact of that mutation on the protein structure and function. The GFP protein can be used as a dramatic tool to visually demonstrate the effect of pivotal amino acid changes on the structure and function of a protein.

BACTERIAL TRANSFORMATION AND OVEREXPRESSION OF TRANSFORMED GENE

Bacterial transformation is of central importance in molecular biology. Transformation is the process by which a bacterium takes up and expresses exogenous DNA, resulting in a newly acquired genetic trait that is stable and heritable. This exogenous DNA can be recombinant DNA molecules that have been constructed in vitro, as well as natural DNA molecules. Transfor-mation is also of historical importance since it led to the discovery by Oswald Avery, in 1944, that DNA was the genetic material. In that historical experi-ment, Avery and colleagues purifi ed DNA from a lethal strain of Streptococ-cus pneumoniae, removing all protein from the DNA. This DNA was then transformed into a harmless strain of the same organism. Injection of the transformed, formerly harmless, strain into mice resulted in their death.

Quick ReferenceAbbreviations

GFP Green fl uorescent protein

pGFP Plasmid for GFP expression

gfp Gene for green fl uorescent protein


6



EDVO-Kit #B

ackg

rou

nd

Info

rmat

ion


For transformation to occur, bacterial cells must be in a particular physiologi-cal state, referred to as competency, in which the bacterial cell wall is made permeable to macromolecules such as DNA. Competency can occur naturally in certain species of Haemophilus and Bacillus when the levels of nutrients and oxygen are low. Competent Haemophilus cells express a membrane-associated transport complex that binds and transfers certain DNA molecules from the medium into the cell where they are then integrated into the bac-terial chromosome and expressed. In nature, the source of the external DNA is from other cells that have died and their cell walls lysed to release their DNA into the surrounding medium.

Much current research in molecular biology involves the transformation of E. coli, an organism that does not naturally enter a state of competency. E. coli can artifi cially be made competent when treated with chloride salts of the metal cations calcium, magnesium and rubidium. In addition, abrupt tran-sitioning between heat and cold can induce competency. It is believed that metal ions and temperature changes affect the structure and permeability of the cell wall and membrane, allowing DNA molecules to pass through. Due to their unstable cell walls, competent E. coli cells are fragile and therefore must be treated carefully.

The number of cells transformed per 1 microgram (µg) of DNA is known as the transformation effi ciency. In practice, much smaller amounts of DNA are used (5 to 100 nanograms, ng) since excessive DNA (>100 ng) inhibits the transformation process. For example, say 10 nanograms (0.01 microgram) of DNA was used to transform cells that were in a fi nal volume of 1 ml. Assume 0.1 ml (100 µg) of these cells were plated on agar medium such that only the cells that acquired the foreign DNA could grow. This procedure is called selection. After incubation (in this example) 100 colonies were found on the

plate. Realizing that each colony origi-nally grew from one transformed cell, the transformation effi ciency in this example is 105 (outlined in Figure 1). In research laboratories, transforma-tion effi ciencies generally range from 1 x 105 to 1 x 108 cells per microgram of DNA. Special procedures can produce cells having transformation effi ciencies approaching 1010.

Transformation is never 100% ef-fi cient. Approximately one in every 10,000 cells successfully incorporates exogenous DNA. However, based on the large number of cells in an average sample (typically 1 x 109), only a small number must be transformed to obtain visible colonies on an agar plate.





Number oftransformants

per μg =

100 transformants

0.01 μg

Specifi c example:

X

fi nal vol at recovery (ml)vol plated (ml)

X

1 ml0.1 ml

=

100,000 (1 x 105)

transformants per μg

Number of transformants μg of DNA

Figure 1:Bacterial Transformation Effi ciency Calculation


303EDVO-Kit #



Backg

rou

nd

Info

rmatio

n


This concept can be demonstrated by plating the same volume of recovered cells on selective and nonselective agar medium. The nonselective bacte-rial agar plates will be covered heavily with untransformed cells, forming a “lawn”, in contrast to individual colonies obtained on the selective agar plate. Transformed cells will grow on selective medium that contains an antibiotic.

Plasmids are usually used to ferry foreign genes into bacteria. They are self-replicating extrachromosomal, double-stranded circular DNA molecules found in many strains of bacteria. Many plasmids contain genes that pro-vide resistance to various antibiotics, including tetracycline, kanamycin, and ampicillin (amp). Ampicillin is a derivative of penicillin that inhibits bacterial growth by interfering with the synthesis of bacterial cell walls. The product of the ampicillin resistance gene is the enzyme β-lactamase. This enzyme is secreted by transformed cells into the surrounding medium, where it destroys ampicillin. Due to this extracellular secretion, cells that are not transformed are able to undergo limited growth in the zones surrounding

Bacterial chromosomal DNA

Supercoiled pGFP DNA

Green FluorescenProtein(GFP)

Figure 3: Overview of transformation and protein expression.

Supercoiled Relaxed

Figure 2: Supercoiled and circular forms of plasmid DNAs

transformed, antibiotic-resistant cells. Colonies consisting of these untrans-formed cells are called "satellites", since they only appear around larger colonies of transformed cells. Larger plating volumes and longer incubation times increase the number of satellite colonies.

Plasmids naturally exist as supercoiled molecules. The two strands of DNA in the supercoiled molecule wind around each other to produce a condensed, entangled structure when compared to relaxed (non-supercoiled) DNA (Fig-ure 2). Competent E. coli cells are sensitive to the conformation of the DNA they will accept. Supercoiled DNA gives the highest transformation effi cien-cies.


8



EDVO-Kit #Th

e Ex

per

imen

t


OVERVIEW OF THE GFP EXPRESSION SYSTEM

In this experiment, the goal is to express the fl uorescent protein (GFP) in transformed bacterial cells (Figure 3, page 7). To begin this process, there must be a means of "turning on" the cloned gfp gene in the recombinant plasmid. In order to have an "off/on" switch for controlling expression, the gene is placed under the control of a DNA sequence known as a "promoter".

A promoter is a sequence of DNA that typically occurs just in front ("up-stream") of the DNA coding sequence (the sequence that specifi es the amino acid sequence for a protein). The chromosome of the host bacterial strain used in this experiment has been genetically engineered to contain the gene for RNA polymerase, which is under control of the lac promoter, and can be turned on (induced) by the presence of a small molecule called IPTG (isopro-pyl-beta-D-thiogalactopyranoside). IPTG binds to and inactivates an inhibi-tor protein known as the lac repressor.

The sequence of events required to turn on expression of gfp is as follows: • Cells are grown in the presence of IPTG (to turn on the lac promoter)

which binds and releases the bound lac repressor. The release of the repressor (inhibitor) allows the RNA polymerase to be produced from the E. coli genome.

• The RNA polymerase, in turn, recognizes the promoter on the plasmid enabling production of large quantities of the fl uorescent GFP protein.

• In summary, a strong promoter, combined with an active RNA poly-merase, allows for very high levels of gfp mRNA (and thus GFP protein expression) in the transformed cells.

Purifi cation of Green Fluorescent Protein by column chromatography and analysis on denaturing polyacrylamide gels.

Bacterial plates from the transformation experiment will be saved and used as seed cultures for growing cells for GFP purifi cation. Cells will be lysed and the bacterial extract containing GFP will be fractionated by chromatography using a molecular sieve matrix. Factors that affect separation include charge, size, shape and associated non-protein biologicals such as carbohydrate residues. The fl uorescent GFP protein will be detected on the column and subsequently in the test tubes by examination under long U.V. light. Esti-mation of protein polypeptide composition and size will be determined by analysis on denaturing polyacrylamide gel electrophoresis.






303EDVO-Kit #



The Exp

erimen

t

BEFORE YOU START THE EXPERIMENT

1. Read all instructions before starting the experiment.

2. Write a hypothesis that refl ects the experiment and predict experimen-tal outcomes.

EXPERIMENT OBJECTIVE:

The objective of this set of experiments is to develop an understanding of bacterial transformation by pGFP plasmid DNA, and purifi cation and charac-terization of the recombinant protein.

BRIEF DESCRIPTION OF EXPERIMENT:

In this experiment, you will transform a strain of competent E. coli that has no antibiotic resistance with supercoiled pGFP plasmid DNA that also has the gene for antibiotic resistance. The plasmid pGFP also codes for the green fl uorescent protein (GFP).

Bacterial cells will be selected for the presence of plasmid by plating them on agar medium containing ampicillin. Only bacterial cells that are transformed with the plasmid will survive selection on ampicillin agar plates and will pro-duce green fl uorescent colonies which will be visible under long wave U.V. Light. The transformation effi ciency will then be estimated.

Transformed cells will be grown on bacterial plates, harvested and lysed. GFP will be purifi ed by column chromatography and analyzed on denaturing polyacrylamide gels. The following are the four modules to this experiment:

I. Transformation of host by pGFP plasmid.II. Plating of transformed cells for GFP purifi cationIII. Purifi cation of GFP by chromatographyIV. Analysis of GFP by denaturing SDS-polyacrylamide gel analysis

Experiment Overview and General Instructions


10



EDVO-Kit #Th

e Ex

per

imen

t

+

Incubateovernight in 37°Cincubation oven

Incubateovernight in 37°Cincubation oven

250 µlCaCl2 / cell

Incubate on ice for 15 minutes

Incubate at 42•Cfor 90 seconds

Incubate on ice for 2 minutes

Add 250 µlLuria Recovery Broth

37°C for 30 minutes

Plasmid DNA

pFluoroGreen™

Transfer 8-10 colonies to the500 µl CaCl2 tube and completely resuspend.

Transfer 250 µl CaCl2 / cell suspension to each tube.

LONG WAVE U.V. LIGHT IS REQUIRED TO OBSERVE

FLUORESCENT COLONIES.

(-) DNA/(-) Amp

(-) DNA/(-) Amp

(-) DNA/(+) Amp

(-) DNA/(+) Amp

(+) DNA/(-) Amp

(+) DNA/(-) Amp

(+) DNA/(+) Amp

(+) DNA/(+) Amp

10 µl

+ D

NA

- D

NA

- D

NA

+ D

NA

CaC

l 2

Control Experiment

E. coli Cells

Prepare 5 Source Plates

DAY BEFORE LAB

500 µl CaCl2

LB Agar

Incubate source plates 16-18hours overnight @ 37° C.

Transfer 50-75 µl cells to eachsource plate and streak

for isolated colonies.

Incubate cells30-60 minutes at 37° C.

Add 2 mlreconstitution

medium to FluoroCell™

vial.

Experiment Overview and General Instructions

MODULE 1: TRANSFORMATION


303EDVO-Kit #



The Exp

erimen

t

Laboratory Safety

Important READ ME!

Transformation experiments contain antibiotics which are used for the selec-tion of transformed bacteria. Students who have allergies to antibiotics such as penicillin, ampicillin, kanamycin or tetracycine should participate in this experiment at their own discretion.

1. Gloves and goggles should be worn routinely as good laboratory practice.

2. Exercise extreme caution when working with equipment which is used in conjunction with the heating and/or melting of reagents.

3. Do not mouth pipet reagents - use pipet pumps or bulbs.

4. The E. coli bacteria used in this experiment is not considered pathogenic. Although it is rarely associated with any illness in healthy individuals, it is good practice to follow simple safety guidelines in handling and disposal of materials contaminated with bacteria.

5. Properly dispose materials after completing the experiment:

A. Wipe down the lab bench with a 10% bleach solution or a laboratory disinfectant.

B. All materials, including petri plates, pipets, transfer pipets, loops and tubes, that come in contact with bacteria should be disinfected be-fore disposal in the garbage. Disinfect materials as soon as possible after use in one of the following ways:

• Autoclave at 121° C for 20 minutes. Tape several petri plates together and close tube caps before

disposal. Collect all contaminated materials in an autoclavable, disposable bag. Seal the bag and place it in a metal tray to pre-vent any possibility of liquid medium or agar from spilling into the sterilizer chamber.

• Soak in 10% bleach solution. Immerse petri plates, open tubes and other contaminated ma-

terials into a tub containing a 10% bleach solution. Soak the materials overnight and then discard. Wear gloves and goggles when working with bleach.

6. Wear gloves, and at the end of the experiment, wash hands thoroughly with soap and water.


12



EDVO-Kit #Th

e Ex

per

imen

t

Module I: Transformation of E. coli with pGFP

NOTE:

Avoid scraping up agar when transferring the cells from the source plate to the tubes with calcium chloride solution. It is important that the cells are resus-pended in the calcium chloride solution and is not left on the tooth-pick or on the wall of the tube.

SETTING UP THE TRANSFORMATION AND CONTROL EXPERIMENT

1. Label one microcentrifuge tube "+ DNA". This will be the transforma-tion tube with plasmid DNA.

2. Label a second microcentrifuge tube "– DNA" . This will be the experi-mental control tube without plasmid DNA.

3. Using a sterile 1 ml pipet, add 0.5 ml of ice cold 0.05 M CaCl2 solution into the ”– DNA” tube and place on ice.

4. With a sterile loop, transfer a group of 8-10 single, well-isolated colonies from the plate labelled E. coli source plate to the “– DNA” tube. Twist the loop vigorously between your fi ngers to dislodge the cells. Vortex the cells to mix and fully suspend the cells in the CaCl2 .

5. Transfer 250 µl of this cell suspension to the tube labeled “+ DNA”.

6. Place both the “– DNA”and the “+ DNA”tubes on ice. At this point, each tube should have 250 µl of the CaCl2 suspended cells.

7. To the tube labeled "+ DNA", add the following:

• 10 µl of pGFP (from tube labeled "pGFP")

8. Incubate the two tubes on ice for 15 minutes.

9. Place both transformation tubes at 42° C for 90 seconds.

This heat shock step facilitates the entry of DNA in bacterial cells.

10. Return both tubes immediately to the ice bucket and incubate for two (2) minutes.

+ D

NA

- D

NA

- D

NA

+ D

NA

- D

NA

+ D

NA

- D

NA

+ D

NA

NOTE:

Remember to resuspend the cells thoroughly by vortexing or vigorous mixing by hand (fl icking the tube of cells). It is very important that the cell suspension is ho-mogenous and no clumps are visible. The cell suspension must appear somewhat cloudy.


303EDVO-Kit #



The Exp

erimen

t

11. Using a sterile pipet, add 250 µl of Luria Recovery Broth to each tube and mix.

12. Incubate the cells for 30 minutes in a 37° C waterbath for a recovery period.

13. While the tubes are incubating, label 4 agar plates as indicated below. Write on the bottom or side of the petri plate.

• Label one unstriped plate: (-) DNA/(-) Amp • Label one unstriped plate (+) DNA/(-) Amp • Label one striped plate: (-) DNA/(+) Amp • Label one striped plate: (+) DNA/(+) Amp • Put your initials or group number on all the plates.

14. After the recovery period, remove the tubes from the water bath and place them on the lab bench. Proceed to plate the cells for incubation.

+ D

NA

- D

NA

- D

NA

+ D

NA


Quick Reference:

DNA and competent cells are combined in a suspen-sion. After the cells have incubated with the DNA, growth medium (recovery broth) is added. Bacterial cells continue to grow through the recovery process, during which time the cell wall is repaired. Cells recover and begin to express the antibiotic resistance gene.

- D

NA

+ D

NA

NOTE: Ensure the entire plate has been completely streaked over with the inoculating loop.

PLATING THE CELLS

Plating cells from the tube labeled "- DNA" (Control Experiment):

15. Use a sterile 1 ml pipet to transfer recovered cells from the tube labeled " – DNA " to the middle of the following plates:

0.25 ml

• 0.25 ml to the plate labeled (-) DNA/(-) Amp• 0.25 ml to the plate labeled (-) DNA/(+) Amp

16. Spread the cells over the entire plate with a sterile inoculating loop (see Figure at left).

17. Cover both plates and allow the liquid to be absorbed.

To avoid contamination when plating, do not set the lid down on the lab bench -- Lift the lid of the plate only enough to allow spreading. Be careful to avoid gouging the loop into the agar.


14



EDVO-Kit #Th

e Ex

per

imen

t

Plating cells from the tube labeled "+ DNA"

18. Use a sterile 1 ml pipet to transfer recovered cells from the tube labeled "+ DNA" to the middle of the following plates:

• 0.25 ml to the plate labeled (+) DNA/(-) Amp • 0.25 ml to the plate labeled (+) DNA/(+) Amp 19. Spread the cells with a sterile inoculating loop in the same manner as

step 16 . 20. Cover the plate and allow the liquid to be absorbed (approximately 15-20 minutes).

PREPARING PLATES FOR INCUBATION

21. Stack your group’s set of plates on top of one another and tape them together. Put your initials or group number on the taped set of plates.

The plates should be left in the upright position to allow the cell suspen-sion to be absorbed by the agar.

22. Place the set of plates in a safe place designated by your instructor.

23. After the cell suspension is absorbed by the agar, you or your instructor will place the plates in the inverted position (agar side on top) in a 37° C bacterial incubation oven for overnight incubation (15-20 hours).

If the cells have not been absorbed into the medium, it is best to incubate the plates upright. The plates are inverted to prevent condensation on the lid, which could drip onto the culture and may interfere with experimental results.

VIEWING PLATES AFTER INCUBATION

24. Darken the room and use a long wave U.V. light to visualize the trans-formed cells that will glow green due to the expression of the green fl uorescent protein.

To visualize the fl uorescent colonies, the long wave U.V. light (EDVOTEK cat. # 969 recommended) can be held underneath the plates in a darkened room.

25. Determine the transformation effi ciency (see next page) and proceed to Module II: Isolation of GFP.

Reminder:

Follow proper procedures for disposal of contaminated materials.

Module 1: Transformation of E. coli with pGFP

Gro

up

4

WEAR SAFETY GOGGLES

Do not use short U.V. light, which can cause burns and serious damage to the eyes.

Important:

Do not allow the plates to incubate for longer than 20 hours at 37° C.

IPTG induced ex-pression in the cells is very high and cell lysis can occur with extended incuba-tion time resulting in low GFP yields.


303EDVO-Kit #



The Exp

erimen

t

DETERMINATION OF TRANSFORMATION EFFICIENCY

Transformation effi ciency is a quantitative determination of how many cells were transformed per 1 µg of plasmid DNA. In essence, it is an indi-cator of how well the transformation experiment worked.

You will calculate the transformation effi ciency from the data you col-lect from your experiment.

1. Count the number of colonies on the plate with ampicillin that is labeled:

(+) DNA/(+) Amp

A convenient method to keep track of counted colonies is to mark the colony with a lab marking pen on the outside of the plate.

2. Determine the transformation effi ciency using the formula:

x =

fi nal vol at recovery (ml)

vol plated (ml)

Number of transformants

per μg

Number of transformants

μg of DNA

Example:Assume you observed 40 colonies:

40transformants

0.05 μg

1600(1.6 x 103)

transformants per μg

x 0.5 ml

0.25 ml=

Module 1: Transformation of E. coli with pGFP

Quick Reference for Expt. 303

50 ng (0.05 μg) of DNA is used.

The fi nal volume at recovery is 0.50 ml. The volume plated is 0.25 ml.


16



EDVO-Kit #Th

e Ex

per

imen

t

A. PLATING OF TRANSFORMED CELLS

1. Obtain two plates of ReadyPour (LB) medium contain-ing AMP/ IPTG.

2. Use your transformation plate (results plate from Module I) as the source of your seed culture. If needed share a plate with another group.

3. With an inoculating loop pick 4-5 isolated GFP express-ing (glowing) colonies.

4. Prepare a smear with the cells using the inoculating loop.

5. Spread plate the cells over the entire LB/Amp/IPTG plate as shown in the fi gure on the left.

6. Repeat steps 3-5 for the second plate.

7. Replace cover onto the plates.

8. Incubate the set of plates in a 37° C incubator over-night for approximately 16-19 hours.

9. Check to make sure there is a confl uent lawn of growth on at least one of the plates.

After 16-19 hour incubation, proceed to Isolation of GFP as described below.Important:

Do not allow the plates to incubate longer than 20 hours at 37° C.

IPTG induced ex-pression in our cells is very high and cell lysis can occur at extended incubation time.

Module II: Isolation of GFP

B. ISOLATION OF GFP

1. Add 500 µl of Tris, EDTA, Glucose (TEG) buffer to a 1.5 ml microcentri-fuge tube. Label the tube with your initials.

2. Select a plate showing the highest GFP expression (maximum glow) after overnight (16-19 hrs) of incubation at 37° C.

3. Using a sterile loop, scrape the entire cell growth from the pGFP plate (from step 2). Twirl the loop containing the colonies in the tube contain-ing TEG buffer. Twirl vigorously and be sure the cells are dislodged into the buffer.

4. Briefl y vortex the tube at maximum speed (5 pulses for 1 second each). Vortex cell mass until cells are thoroughly resuspended.

5. Repeat steps 1-4 with the other plate.

NOTE: Ensure the entire plate has been completely streaked over with the inoculating loop.

Step 1

Step 2 Step 3

Step 4 Step 5


303EDVO-Kit #



The Exp

erimen

t

6. Place your microcentrifuge tube containing the GFP cells in the -20° C freezer for 15 minutes, or until frozen. Lay the tube on its side to ensure rapid freezing.

7. After GFP cells are completely frozen, remove the microcentrifuge tube from the freezer, and put it in a 37° C waterbath to thaw the cells.

8. Repeat steps 6 – 7 two more times. (Freezing and thawing will help lyse the cells.)

9. Centrifuge the tube in a microcentrifuge for 10 minutes at maximum speed.

Note: Make sure you have balanced your tube before starting the micro-centrifuge!

10. At this point, the supernatant should contain the green fl uorescent pro-tein (it should be bright green).

Note: If the supernatant is not fl uorescent and/or the cell pellet is fl uo-rescent, repeat the freezing/thawing/centrifugation steps (steps 6 - 9) until the green fl uorescent protein (GFP) is released into the supernatant.

11. Transfer 200 µl of “glowing” supernatant to a clean tube and label it “GFP cell extract”. Store the extract and any leftover supernatant in the freezer for use in Module III: Purifi cation of GFP by Column Chromatog-raphy.


Protocol Hint:

Lay the tubes on their sides in the freezer - cells will freeze quicker.

OPTIONAL STOPPING POINT






18



EDVO-Kit #Th

e Ex

per

imen

t

Module III: Purifi cation of GFP by Column Chromatography

PACKING AND EQUILIBRATING THE COLUMN

1. Vertically mount the column on a ring stand. Make sure it is straight.

2. Slide the cap onto the spout at the bottom of the column. Fill about one-third of the column with the elution buffer.

3. Mix the slurry (molecular sieve) thoroughly by swirling or gently stirring.

4. Carefully pipet the mixed slurry into the column by letting it stream down the inside walls of the column.

If the fl ow is stopped by an air pocket, stop adding the slurry and fi rmly tap the column until the air is removed and the slurry continues to fl ow down the side of the column.

5. Place an empty beaker under the column to collect wash buffer.

6. Remove the cap from the bottom of the column and allow the matrix to pack into the column.

7. Wash the packed column with 5 ml of 1x elution buffer. Do not allow the column to dry.

8. Slide the cap onto the spout and make sure it does not drip.

NOTE:

The loading of the col-umn and subsequent elution will be done at room temperature. The elution buffers and the fractions col-lected will be stored on ice as they elute from the column.


Do not allow the column to dry!


303EDVO-Kit #



The Exp

erimen

t


COLLECTING COLUMN FRACTIONS OF (GFP) PROTEIN

1. Label the fi rst set of eight microtest tubes #1-8. Mark the tubes for 0.5 ml volume on the outside using a permanent marker.

2. Slowly load the column with 0.2 ml of the GFP extract. Allow the extract to completely enter the column.

3. Elute the column with 1X elution buffer. • Add buffer slowly (several drops at a time) to avoid diluting the

protein sample. • Using the graduated marks on the sides of the tubes, collect 0. 5

ml fractions in the labeled microcentrifuge tubes. • Store fractions on ice immediately upon collection.

* * * Remember - Do not allow the column to dry. * * *Continue adding 1X elution buffer until all of

the GFP fractions have been collected.

4. Monitor the progress of the GFP in the gel matrix by illuminating the column with the long wave U.V. light source (it may help to dim the lights in the lab/classroom).

5. When the GFP protein band almost reaches the bottom of the column (near the frit), start collecting the fractions in the microtiter plate.

Collect 4 drops per well starting with row 1, column A of the microtiter plate and work your way from left to right (i.e. A1, B1, C1, etc.). When you reach the end of row 1, continue with row 2.

6. Continue monitoring the progress of the GFP in the column and collect fractions until the GFP has been completely eluted.

7. Check the fractions in the microtiter plate by using the long wave U.V. light. Identify two or three wells that contain the brightest levels of fl uorescent proteins.

Note: The tube fractions from step 3 should not contain any GFP.

8. Save the fractions containing the highest levels of GFP proteins for further analysis by SDS gel electrophoresis (optional).

WEAR SAFETY GOGGLES

Monitor the elution of GFP by shining a long U.V. light on the side of the column.

Do not use short U.V. light, which can cause burns and serious damage to the eyes.


If time does not permit you to continue with the SDS polyacrylamide analysis, you may freeze the fractions at -20° C and perform the assays at a later date.


20



EDVO-Kit #Th

e Ex

per

imen

t

SAMPLE PREPARATION FOR DENATURING SDS-GEL ELECTROPHORESIS

1. Select a peak extract (from the microtiter plate) and transfer 30 µl to a clean screw-cap microcentrifuge tube. Label the tube "GFP denatured".

2. In another screw-cap microcentrifuge tube, transfer 30 µl of the peak extract and label the tube "GFP native".

PREPARING NATIVE PROTEIN (UNBOILED)

The protein in its native form can be shown to fl uoresce with a long wave U.V. light.

3. Add 10 µl of 50% glycerol (L) to each of the tubes labeled "GFP native".

4. Mix and set these tubes aside for electrophoresis.

PREPARING DENATURED PROTEINS (BOILED)

5. To denature the protein samples, add 25 µl of protein denaturing solu-tion (K) to each of the tubes labeled "GFP denatured". The denaturing solution contains sodium dodecylsulfate (SDS) and 2-mercaptoethanol.

6. Bring a beaker of water, covered with aluminum foil, to a boil.

7. Make sure the sample tubes to be denatured (boiled) are tightly capped (and thawed if GFP samples have been stored at -20° C).

8. The bottom of the tubes should be pushed through the foil and im-mersed in the boiling water for 5 minutes. The tubes should be kept suspended by the foil.

9. Allow the tubes to cool for a few minutes at room temperature.

10. Proceed with Electrophoresis of Proteins as outline on page 21.

DENATURING THE STANDARD PROTEIN MARKERS

• If your standard protein marker has not been rehydrated and boiled by your instructor, add 130 µl of distilled or deionized water to it and let the sample rehydrate for several minutes. Vortex or mix vigorously. Then, proceed to denature the standard protein markers by boiling for 5 minutes as described above.

• If your standard protein marker has already been denatured, proceed with Electrophoresis of Proteins as outlined on page 21.

Quick Reference:

Proteins unfold and lose their tertiary structures by boiling for 5 min-utes in the presence of denaturing solutions which contain SDS and 2-mercaptoethanol. In the absence of boiling, regions of the protein can remain intact in their native state.

Module IV: Analysis of GFP by Denaturing SDS-Gel Electrophoresis

CAUTION:

Use screw-cap tubes for boiling denatured samples.


303EDVO-Kit #



The Exp

erimen

t


PREPARING THE POLYACRYLAMIDE GEL FOR ELECTROPHORESIS

Precast Polyacrylamide Gels:

Precast polyacrylamide gels will vary slightly in design. Procedures for their use will be similar.

1. Open the pouch containing the gel cassette with scissors. Remove the cassette and place it on the bench top with the front facing up.

Note: The front plate is smaller (shorter) than the back plate.

2. Some cassettes will have tape at the bottom of the front plate. Remove all of the tape to expose the bottom of the gel to allow electrical con-tact.

3. Insert the Gel Cassette into the electrophoresis chamber.

4. Remove the comb by placing your thumbs on the ridges and pushing (pressing) upwards, carefully and slowly.

Wear gloves and safety goggles

PROPER ORIENTATION OF THE GEL IN THE ELECTROPHORESIS UNIT

1. Place the gel cassette in the electrophoresis unit in the proper orientation. Protein sam-ples will not separate in the gel if the cassette is not oriented correctly. Follow the directions accompanying the specifi c apparatus.

2. Add the diluted buffer into the chamber. The sample wells and the back plate of the gel cas-sette should be submerged under buffer.

3. Rinse each well by squirting electrophoresis buffer into the wells using a transfer pipet.

The gel is now ready for practice gel loading or sample loading.

A polyacrylamide gel cassette in the EDVOTEK® Vertical Electrophoresis Apparatus, Model #MV10.

Negative ElectrodeBlack Dot (-)

Buffer Level

Sample Well

Long Cassette Plate

Polyacrylamide Gel

Viewing Level(cut out area on front support

panel)

Platinum wire

Short Cassette Plate

Gel Cassette Clip

Micropipet withFine tip


22



EDVO-Kit #Th

e Ex

per

imen

t

PRACTICE GEL LOADING (OPTIONAL)

EDVOTEK® Cat. #638, Fine Tip Micropipet Tips are recommended for loading samples into polyacrylamide gels. A regular micropipet tip may damage the cassette and result in the loss of protein samples.

1. Place a fresh fi ne tip on the micropipet. Aspirate 20 µl of practice gel loading solution.

2. Place the lower portion of the fi ne pipet tip between the two glass plates, below the surface of the electrode buffer, directly over a sample well. The tip should be at an angle pointed towards the well. The tip should be partially against the back plate of the gel cassette but the tip opening should be over the sample well, as illustrated in the fi gure on the previous page.

Do not try to jam the pipet tip in between the plates of the gel cassette.

4. Eject all the sample by steadily pressing down on the plunger of the automatic pipet.

Do not release the plunger before all the sample is ejected. Premature release of the plunger will cause buffer to mix with sample in the micro-pipet tip. Release the pipet plunger after the sample has been delivered and the pipet tip is out of the buffer.

5. Before loading protein samples for the actual experiment, the practice gel loading solution must be removed from the sample wells.

Do this by fi lling a transfer pipet with buffer and squirting a stream into the sample wells. This will displace the practice gel loading solution, which will be diluted into the buffer and will not interfere with the experiment.



303EDVO-Kit #



The Exp

erimen

t

LOADING PROTEIN SAMPLES

Change pipet tips between loading each sample. Make sure the wells are cleared of all practice loading solution. Gently squirt electrophoresis buffer into the wells with a transfer pipet.

Two student groups can share a gel. Some of the samples contain denatur-ing solution which contains SDS and 2-mercaptoethanol. The samples should be loaded in the following manner:

First Student Group

Lane 1 20 µl of standard protein markers (boiled for 5 min)Lane 2 20 µl of GFP native #1 (not boiled)Lane 3 20 µl of GFP denatured #1 (boiled for 5 min)Lane 4 20 µl of GFP native #2 (not boiled)Lane 5 20 µl of GFP denatured #2 (boiled for 5 min) Second Student Group

Lane 6 20 µl of standard protein markers (boiled for 5 min)Lane 7 20 µl of GFP native #1 (not boiled)Lane 8 20 µl of GFP denatured #1 (boiled for 5 min)Lane 9 20 µl of GFP native #2 (not boiled)Lane 10 20 µl of GFP denatured #2 (boiled for 5 min)

RUNNING THE GEL

1. After the samples are loaded, carefully snap the cover all the way down onto the electrode terminals. On EDVOTEK® electrophoresis units, the black plug in the cover should be on the terminal with the black dot.

2. Insert the plug of the black wire into the black input of the power sup-ply (negative input). Insert the plug of the red wire into the red input of the power supply (positive input).

Volts Recommended Time

Minimum Optimal

125 60 min 75 min

Time and Voltage

3. Set the power supply at the required voltage and run the electrophoresis for the length of time as determined by your instructor. When the current is fl owing, you should see bubbles forming on the electrodes. The sudsing is due to the SDS in the buffer.

4. After the electrophoresis is fi nished, turn off power, unplug the unit, disconnect the leads and remove the cover.

EDVOTEK® Cat. #638, Fine Tip Micropipet Tips are recommended for loading samples into polyacrylamide gels. A regular micropipet tip may damage the cassette and result in the loss of protein samples.

NOTE:

Shine the long wave U.V. light on the gel while the native proteins are separating.



24



EDVO-Kit #Th

e Ex

per

imen

t

NOTES:

Protein polyacrylamide gels can be stained with Protein InstaStain® cards in one easy step. Gloves must be worn during this procedure. Avoid touching the blue side of the paper with-out gloves, as this will affect its ability to stain the gel uniformly.

Polyacrylamide gels are very thin and fragile. Use care in handling to avoid tearing the gel.


1. After electrophoresis, lay the cassette down and remove the front plate by placing a thin spatula at the top edge, near the sample wells, and gently lifting it away from the larger back plate. In most cases, the gel will stay on the back plate. If it partially pulls away with the front plate, let it fall onto the back plate. Handle very carefully as the thin gels are extremely fragile.

2. Transfer gel with on the back plate to a clean tray.

3. Add a suffi cient volume (approximately 100 ml) of the fi xative solution into the tray to cover the gel and back plate. (Use enough solution to cover the gel.)

4. Remove the back plate from the tray, leaving just the gel in the tray containing the fi xative solution.

If the gel sticks to the plate, pipet some of prepared fi xative solution onto the gel and gently nudge the gel off the plate.

5. Gently fl oat a sheet of Protein InstaStain® with the stain side (blue side down) in the fi xative liquid. Cover the gel with plastic wrap to prevent evaporation.

6. Allow the Protein InstaStain® paper to stain gel for about an hour at room temperature with occasional or continuous agitation.

7. Remove the paper after an hour and allow the gel to gently agitate on a rocking platform or just on the lab bench for 1-3 hours or overnight.

Overnight staining of protein gels yields a more optimal result. Pour off the staining solution from step 7 the following day and add fresh destaining fi xative solution to cover the gel.

8. After staining, Protein bands will appear medium to dark blue against a light background* and will be ready for excellent photographic results.

NO DESTAINING IS REQUIRED.

*If the gel is too dark, destain at room temperature with continuous agita-tion in several changes of fresh fi xative solution until the appearance and contrast of the protein bands against the background improve.


303EDVO-Kit #



The Exp

erimen

t

STORING THE GEL

• Gel may be left in deionized water for a longer storage time with no loss in sensitivity and band intensity. This step should be performed once a desired background and stained protein bands are is obtained. Pour off the destaining solution from step 7 and add a suffi cient amount of deionized water to cover the gel. Wash with water for a minimum of 1 hour or until the desired background clarity is obtained.

• For permanent storage, the gel can be dried between two sheets of cel-lophane (saran wrap) stretched in an embroidery hoop. Air dry the gel for several days until the gel is paper thin. Cut the “extra” saran wrap surrounding the dried gel. Place the dried gel overnight between two heavy books to avoid curling. Tape it into a laboratory book.



26



EDVO-Kit #Th

e Ex

per

imen

t

DETERMINATION OF MOLECULAR WEIGHTS

If measurements are taken directly from the gel, skip steps 1 and 2.

1. Take a transparent sheet, such as cellulose acetate (commonly used with overhead projectors) and lay it over the wrapped gel.

2. With a felt-tip pen, carefully trace the outlines of the sample wells. Then trace over all the protein bands on the gel.

In the example shown above, the standard molecular weights are: 94,000 30,000 67,000 20,000 38,000 14,000

8

10

76

5

4

3

2

1

9

876

5

4

3

2

1

9

876

5

4

3

2

1

9

100,000

10,000

6 7 8 9 10

Centimeters

Mol

ecul

ar W

eigh

t

Phosphorylase

Bovine Serum Albumin

Ovalbumin

Carbonic anhydrase

5

Soybean Trypsin Inhibitor

Lysozyme

3. Measure the migration distance, in centimeters (to the nearest millimeter) of every major band in the gel. (Ig-nore the faint bands, refer to Idealized Schematic.) All measurements should be from the bottom of the sample well to the bottom of the protein band.

4. Using semilog graph paper, plot the migration distance or Rf of each stan-dard protein on the non-logarithmic x-axis versus its molecular weight on the logarithmic y-axis. Choose your scales so that the data points are well spread out. Assume the second cycle on the y-axis represents 10,000 to 100,000 (see example at left).

5. Draw the best average straight line through all the points. This line should roughly have an equal number of points scattered on each side of the line. As an example, refer to the fi gure at left. This method is a linear approximation.

6. Using your standard graph, determine the molecular weight of the three unknown proteins. This can be done by fi nding the Rf (or migration distance) of the unknown band on the

x-axis and drawing a straight vertical until the standard line is intersected.

A straight line is then made from the intersection across to the y-axis where the approximate molecular weight can be determined.



303EDVO-Kit #



The Exp

erimen

t

8,000

10,000

7,000

6,000

5,000

4,000

3,000

2,000

9,000

80 70

60

50

40

30

20

10

90 100

1,000

800 700

600

500

400

300

200

900

X-axis: Migration distance (cm)

1 cm 2 cm 3 cm 4 cm 5 cm 6 cm

Y-a

xis:

Mo

lec

ula

r We

ight


28



EDVO-Kit #Th

e Ex

per

imen

t

Answer the following study questions in your laboratory notebook or on a separate worksheet.

1. Exogenous DNA does not passively enter E. coli cells that are not compe-tent. What treatment do cells require to be competent?

2. Why did the recovery broth used in this experiment not contain ampicil-lin?

3. What evidence do you have that transformation was successful?

4. What is the purpose of the three control plates?

5. What is the source of the GFP fl uorescence?

6. Why is the molecular sieving matrix swelled prior to packing the col-umn?

7. What is the basis of molecular sieve chromatography?

Study Questions

Exploring Biotechnology with the Green Fluorescent Protein 29

EDVOTEK - The Biotechnology Education Company® 1.800.EDVOTEK • www.edvotek.com

FAX: 202.370.1501 • email: [email protected]

303EDVO-Kit #


Instructor’s Guide

IMPORTANT READ ME!

Transformation experiments contain antibiotics which are used for the selec-tion of transformed bacteria. Students who have allergies to antibiotics such as penicillin, ampicillin, kanamycin or tetracycine should not participate in this experiment.

ORGANIZING AND IMPLEMENTING THE EXPERIMENT

Class size, length of laboratory sessions, and availability of equipment are factors which must be considered in the planning and the implementation of this experiment with your students.

The guidelines that are presented in this manual are based on ten laboratory groups consisting of two, or up to four students. The following are imple-mentation guidelines, which can be adapted to fi t your specifi c set of cir-cumstances. If you do not fi nd the answers to your questions in this section, a variety of resources are available at the EDVOTEK web site. In addition, Technical Service is available from 9:00 am to 6:00 pm, Eastern time zone. Call 1-800-EDVOTEK for help from our knowledgeable technical staff.

Technical ServiceDepartment

FAX: 202.370.1501web: www.edvotek.com

email: [email protected]

Mon - Fri9:00 am to 6:00 pm ET

Mon - Fri 9 am - 6 pm ET

(1-800-338-6835)

EDVO-TECH SERVICE

1-800-EDVOTEK

Please have the following information:

• The experiment number and title• Kit Lot number on box or tube • The literature version number (in lower right corner)• Approximate purchase date

Visit our web site for information about

EDVOTEK's complete line of experiments for

biotechnology and biology education.

Online Ordering now available

Note: Polyacrylamide gels are not included. You may choose to purchase precast gels (Cat. #s 651 or 652).


30



EDVO-Kit #In

stru

cto

r’s

Gu

ide

Notes to the Instructor:

NATIONAL CONTENT AND SKILL STANDARDS

By performing this experiment, students will develop skills necessary to do scientifi c inquiry, learn new techniques using several types of biotechnol-ogy equipment, and will learn standard procedures used in transformation. Analysis of the experiments will provide students the means to transform an abstract concept into a concrete explanation.

SUGGESTED IMPLEMENTATION SCHEDULE

Note: Overnight incubations are necessary for certain steps. Multiple steps can be performed in one day. Prior to the Lab

Day 1:

• Prepare agar plates • Prepare E. coli Cells (overnight incubation). • Dispense the DNA and control buffer


Day 2: (Day of Lab Experiment)

• Equilibrate water baths at 37° C and 42° C • Equilibrate incubation oven at 37° C • Students transform cells and plate for overnight incubation.

Day 3: (Day after Lab Experiment)

• Students observe transformants and controls • Students calculate transformation effi ciency • Follow clean up and disposal procedures as outlined in the Labora-

tory Safety section. • Students save the plates with GFP transformants for Module II


Day 4

• Plating of Transformed Cells for GFP Purifi cation

Day 5

• Harvesting of Transformed Cells • Preparation of Cell Lysis • Preparation of Column for Chromatography


303EDVO-Kit #



Instru

ctor’s G

uid

e

Notes to the Instructor:


Day 6

• Purifi cation of GFP by Column Chromatography


Day 7

• Analysis of GFP by Denaturing SDS-Polyacrylamide Gels

APPROXIMATE TIME REQUIREMENTS

Modules I and II:

1. Preparation of E. coli: plate for individual colonies and incubate at 37° C for 16 to 24 hours before the laboratory (overnight incubation).

2. Preparation of agar plates: plates can be prepared several days in ad-vance and stored inverted (agar side on top) in the refrigerator. Prepa-ration requires approximately 1 hour.

3. Dispensing the DNA and control buffer: This can be done the day be-fore the lab and stored in the refrigerator. Requires approximately 15 minutes.

4. Equilibration of equipment: On the day of the experiment, allow ample time for the equilibration of the water baths at 37° C and 42° C and a bacterial incubation oven at 37° C .

5. Transformation and plating: Each group will perform the transforma-tion experiment and plate four sets of bacterial cells. These procedures require approximately 50 minutes.

6. Overnight incubation: Incubate plates approximately 15-20 hours at 37° C. Additional colonies will also appear between 24 - 48 hours at

room temperature.

Modules III and IV:

1. Preparation of the slurry: The slurry must hydrate for 30-60 minutes/ Dispensing will take only a few minutes. Other materials to assemble will take approximately 15 minutes.

2. Materials for vertical polyacrylamide electrophoresis can be prepared in approximately 15 minutes.


32



EDVO-Kit #In

stru

cto

r’s

Gu

ide

Pre-Lab Preparations - Module I: Transformation of E. coli

POUR AGAR PLATES (Prior to the Lab experiment)

• For optimal results, prepare plates two days prior to plating and set aside the plates inverted at room temperature.

• If they are poured more than two days before use, they should be stored inverted in the refrigerator. Remove the plates from the refrigerator and store inverted for two days at room temperature before use.

Heat the ReadyPour™ Medium

1. Equilibrate a water bath at 60° C for step 5 below.

2. Loosen, but do not remove, the cap on both ReadyPour medium bottle to allow for the venting of steam during heating.

Caution: Failure to loosen the cap prior to heating or microwaving may cause the ReadyPour medium bottle to break or explode.

3. Squeeze and vigorously shake the plastic bottles to break up the solid agar into chunks

4. Heat the ReadyPour medium bottles, one at a time, by one of the methods outlined below. When completely melted, the amber-colored solution should appear free of small particles.

A. Microwave method:

• Heat the bottle on High for two 30 second intervals. • Using a hot glove, swirl and heat on High for an additional 25 sec-

onds, or until all the ReadyPour medium is dissolved. • Using a hot glove, occasionally swirl to expedite melting.

B. Hot plate or burner method:

• Place the bottle in a beaker partially fi lled with water. • Heat the beaker to boiling over a hot plate or burner. • Using a hot glove, occasionally swirl to expedite melting.

5. Allow the melted ReadyPour medium to cool. Placing the bottles in a 60° C water bath will allow the agar to cool, while preventing it from prematurely solidifying.

When the ReadyPour™ medium reaches approximately 60° C, the bottles will be warm to the touch but not burning

hot.

Wear Hot Gloves and Goggles during all steps involving heating.

60˚C

Small bottle of ReadyPour™:

Use for large source plates and small control plates (no amp/ IPTG).

Large bottle of ReadyPour™:

Use for small plates containing ampicillin and IPTG.


303EDVO-Kit #



Instru

ctor’s G

uid

e

Pre-Lab Preparations - Module I: Transformation of E. coli with pGFP

Label (“Stripe”) the Plates

• Open the packets of 60 x15 mm petri plates and stack the plates neatly.

• Use a lab marker to “stripe” the sides of 28 petri plates. These plates

will be used for medium with ampicillin.

• Do not stripe the remaining 12 petri plates. These plates will be the control plates.

Pour the Plates

Note: The small bottle of agar medium will be used to make the 5 source plates and 12 control plates.

1. Pour 5 large E. coli source plates

Use a 10 ml pipet and pipet pump to pour the 5 large plates, 10 ml each, with the ReadyPour medium without ampicillin.

2. Pour 12 control plates (no ampicillin, no-stripe):

Use a fresh 10 ml pipet (or the same pipet from previous step 1) and pipet pump to pour the 12 control plates, 5 ml each with ReadyPour medium without ampicillin or IPTG.

• Use a sterile 10 ml pipet with a pipet pump to transfer the designated volume of medium to each petri plate. Pipet carefully

to avoid forming bubbles.

• Rock the petri plate back and forth to obtain full coverage.

• If the molten medium contains bubbles, they can be removed by passing a fl ame across the surface of the medium.

• Cover the petri plate and allow the medium to solidify.

Quick Reference: Pouring Agar Plates

Note: The large bottle of agar medium will be used to make the LB plates containing Ampicillin and IPTG.

3. Add the IPTG (D) to the large bottle of cooled Ready Pour medium. Re-cap the bottle and swirl to mix the IPTG.

4. Add the entire amount of ampicillin powder (C) to the remaining molten ReadyPour medium in the bottle.

5. Recap the bottle and swirl to completely mix the ampicillin.

Add reagents to medium which has been cooled. Hot medium will cause reagents, such as ampicillin and IPTG, to rapidly decompose.

Small bottle of ReadyPour™:

Use for large source plates and small control plates (no amp/ IPTG).

Large bottle of ReadyPour™:

Use for small plates containing ampicillin and IPTG.


34



EDVO-Kit #In

stru

cto

r’s

Gu

ide

6. Pour 28 plates (with ampicillin & IPTG, striped plates):

Use a fresh 10 ml pipet to pour the remaining 28 striped plates, 5 ml each with ReadyPour medium containing ampicillin and IPTG.

7. Allow the agar to cool and resolidify.

Note: If plates will be used within two days, store in a sealable plastic bag so the plates will not dry out. Store at room temperature, inverted.


Summary of Poured Plates:

5 Source plates - large plates: 10 ml each - ReadyPour medium

12 Control plates - small no stripe plates: 5 ml each - ReadyPour medium without ampicillin or ITPG

28 Plates - small striped plates: 5 ml each - ReadyPour medium with IPTG and ampicillin • 12 of these plates will be used for Module I: Transformation of E. coli with pGFP. • 12 of these plates will be used for Module II: Isolation of GFP. There are 4 extra plates. (Wrap these plates in the original

plastic sleeves and store inverted in the refrigerator until needed.)

Reminder:Follow proper procedures for disposal of contaminated materials.


303EDVO-Kit #



Instru

ctor’s G

uid

e


DAY BEFORE THE LAB

This experiment requires preparation of isolated E. coli host transfor-mation colonies 16-18 hours before the laboratory experiment, so plan accordingly.

Important: Do not prepare source plates more than 18 hours before the experiment. Old source plates will compromise the success of the trans-formation experiment.

PREPARATION OF E. COLI CELLS

1. Use a sterile pipet to add 2 ml of cell reconstitution medium to the vial of FluoroCells™.

2. Replace the rubber stopper and cap. Mix by gently inverting until the freeze dried plug is dissolved.

3. Incubate the vial of cells for 30-60 minutes in a 37° C incubation oven.

Growth should be evident (broth should be slightly turbid or cloudy). If growth is not evident, incubate for a longer period of time.

4. Transfer 50-75 µl of cells to each source plate and streak the cells on one quadrant of each plate with a sterile loop (Step A, fi gure below).

5. With the same loop, streak through the cells once or twice into another clean section of the plate (Step B) to obtain isolated colonies. Streak through the second section once or twice into the remaining clean section of the place (Step C).

6. Label the plates “E. coli”, invert and incubate the plates overnight (16-18 hours) at 37° C in an incubation oven.

If growth on plates is heavy (i.e. few or no isolated colonies), instruct students to touch the toothpick to a small amount of cells. Colonies’ size should be approximately 2-4 mm.

Source PlateE. coli Cells

Source PlateE. coli Cells

LB Agar

Incubate source plates 16-18hours overnight @ 37° C.

Transfer 50-75 µl cells to eachsource plate and streak.

Incubate cells30-60 minutes at 37° C.

Add 2 mlreconstitution

medium to FluoroCell™

vial.

Students beginexperiment

Students transfer5-8 large colonies.

500 µlCaCl2

Primary Smear

Step A - Primary Streak Step B - Secondary Streak

Step C - Tertiary Streak


36



EDVO-Kit #In

stru

cto

r’s

Gu

ide

DAY OF THE LAB:

1. Dispense 1 ml of CaCl2 into microcentrifuge tubes for each of the 10 groups and place on ice.

2. Dispense 1.5 ml of Luria Broth Medium ("Recovery broth") into tubes for each of the 10 groups and keep at room temperature.

Alternatively, the Luria Broth Medium bottle can be placed at a classroom pipeting station for students to share.


Each Group Requires:

• Sharing - one of 5 E. coli source plates• 1 Tube (1 ml) CaCl2• 1 Tube pGFP plasmid DNA• 1 Sterile tube (1.5 ml) "Recovery broth"• 2 Sterile inoculating loops

Classroom Equipment:

• Water bath(s)• Incubation Oven

Preparation of DNA

3. Label 10 tubes "pGFP".

4. Place the tube of supercoiled pGFP on ice.

5. Before dispensing the DNA, tap the tubes until all the sample is at the tapered bottom of the tube.

6. Using an automatic micropipet, dispense 12 µl of supercoiled DNA to each of the microtest tubes labeled "pGFP".

Note: Students will use 10 μl for the transformation experiment.

7. Cap the tubes and place them on ice.

Pre-Lab Preparations - Module II: Isolation of GFP

1. Dispense 1 ml of TEG buffer (G) for each group.

2. Assemble materials for student groups (for use over the course of two days).

• 2 Striped plates• 2 Unstriped plates• 4 Sterile 1 ml pipets

• 2 plates of ReadyPour™ (LB) medium containing AMP/ IPTG• Inoculating loops• 1 ml of TEG buffer (G)• microcentrifuge tubes• micropipets and pipet tips


303EDVO-Kit #



Instru

ctor’s G

uid

e

Pre-Lab Preparations - Module III: Purifi cation of GFP

COLUMN ELUTION BUFFER

1. Dilute the Column Elution Buffer by mixing 30 ml of 10X Column Elution Buffer (H) with 270 ml of Distilled water.

2. Label this buffer "1X Column Elution Buffer".

PREPARATION OF THE MOLECULAR SIEVE MATRIX

3. Hydrate the Dry Molecular Sieve Matrix (J) in 40 ml of 1X Column Elution buffer (diluted H).

4. Gently stir occasionally for a minimum of 30 to 60 minutes.

5. Aliquot 6ml for each of the six groups.

CELL EXTRACT CONTAINING GFP FLUORESCENT PROTEIN (CONTROL)

6. Thaw the frozen GFP extract (F) at room temperature and immediately place on ice.

7. Label 6 tubes "GFP extract". Aliquot 220 µl of the extract into the tubes. Place immediately back on ice. Use this only as a control.

RECONSTITUTION OF LYOPHILIZED PROTEIN MOLECULAR WEIGHT STANDARDS

Once rehydrated, the tube of Protein Molecular Weight Standards (I) con-tains enough material for loading 6 wells. The tube can be boiled in con-junction with the denatured GFP extracts.

8. Add 130 µl of distilled or deionized water to the tube of Protein Molecu-lar Weight Standards (I) and allow the material to hydrate for several minutes. Vortex or mix vigorously.

9. Bring a beaker of water, covered with aluminum foil, to a boil. Remove from heat.

10. Make sure the sample tubes are tightly capped (in screw-cap tubes) and well-labeled. The bottom of the tubes should be pushed through the foil and immersed in boiling water for 5 minutes. The tubes should be kept suspended by the foil.






38



EDVO-Kit #In

stru

cto

r’s

Gu

ide

Pre-Lab Preparations - Module IV: Analysis of GFP by SDS-Electrophoresis

For Partial Purifi cation and Sample Preparation, each group requires:

1 Chromatography column 1 Ring stand with clamp 8 Microtest tubes 1 Microtiter plate, piece 6 ml Molecular Sieve Matrix (hydrated J) 25 ml 1x Column Elution Buffer (diluted H) 220 μl "GFP extract" (F) 75 μl Protein Denaturing Solution (K) 75 μl 50% Glycerol Solution (L) Automatic micropipet and tips 5 ml pipets and pump

20 μl Protein molecular weight marker (to be shared by student groups)

11. The markers can be aliquoted for each of the student groups, or stu-dents can share the rehydrated sample stock tube. Have students load samples onto the polyacrylamide gel while the samples are still warm to avoid aggregation. The volume of sample to load per well is 20 µl.

12. Store any unused portion of reconstituted sample at -20° C and repeat steps 9 and 10 when using samples at a later time.

OTHER COMPONENTS

13. Thaw and place on ice the Protein Denaturing Solution (K) and 50% glycerol. Dispense for each group according to chart below.


303EDVO-Kit #



Instru

ctor’s G

uid

e

PREPARING ELECTROPHORESIS BUFFER

Prepare the electrophoresis buffer by adding and mixing 1 part Tris-Glycine-SDS 10x buffer concentrate to 9 parts distilled water.

The approximate volume of 1X electrophoresis buffer required for ED-VOTEK® Protein Vertical Electrophoresis units are listed in the table below. The buffer should just cover the back plate of the gel cassette.

ELECTROPHORESIS TIME AND VOLTAGE

Your time requirements will dictate the voltage and the length of time for your samples to separate by electrophoresis. Approximate recommended times are listed in the table below.

Conc. (10x) Distilled Total Buffer + Water = Volume

58 ml 522 ml 580 ml

95 ml 855 ml 950 ml

EDVOTEK Model #

MV10

MV20

Tris-Glycine-SDS Electrophoresis Buffer

Pre-Lab Preparations - Module IV: Analysis of GFP by SDS-Electrophoresis

Volts Recommended Time

Minimum Optimal

125 60 min 75 min

Time and Voltage

PREPARING STAINING AND DESTAINING SOLUTIONS

The stock solution is used for staining and destaining with Protein InstaStain®.

1. Solution for staining with Protein InstaStain®

Prepare a stock solution of Methanol and Glacial Acetic Acid by combin-ing 180 ml Methanol, 140 ml Distilled water, and 40 ml Glacial Acetic Acid. Staining of Protein gel(s) is optional.

2. Destaining Solution

Use the stock solution of Methanol, Glacial Acetic Acid and distilled water (in Step 1) to destain the gel(s).


40



EDVO-Kit #In

stru

cto

r’s

Gu

ide

Experiment Results and Analysis

(+) DNA/(+) Ampplated with cells

pGFP

Result: individual colo-nies that will fl uoresce when exposed to long wave U.V. light.

Demonstrates: Transfor-mation of cells resistant to ampicillin due to the uptake of pGFP. Host bacterial cells that are not transformed will not grow in the presence of ampicillin.

(+) DNA/(-) Ampplated with cells

pGFP

Result: white colonies.May look like a smeared layer of cells.

Demonstrates: Un-transformed and trans-formed cells are viable in the absence of ampi-cillin. The majority of the growth are the un-transformed cells and therefore overshadow the transformed fl uo-rescent cells.

(-) DNA/(+) Ampplated with control

cells (no DNA)

Result: No growth

Demonstrates: Cells are sensitive to ampicillin. Without pGFP, they are not ampicillin-resistant.

(-) DNA/(-) Ampplated with control cells

(no DNA)

Result: No fl uorescent cells visible. White colo-nies. May look like a smeared layer of cells.

Demonstrates: Host bac-terial cells are viable in the absence of ampicillin.

Quick Reference

Control experiments:

(-) DNA/(-) Amp Untransformed host bacterial cells (do not contain plasmid DNA) will grow on LB medium.

(-) DNA/(+) Amp Untransformed host bacterial cells (do not contain plasmid DNA) will not grow on LB medium in the presence of ampicillin.

(+) DNA/(-) Amp Both transformed (contain plasmid DNA) and untransformed cells (do not contain plasmid DNA) will grow in the absence of ampicillin.

Positive Results from the transformation experiment:

(+) DNA/(+) Amp Only transformed cells (contain plasmid DNA) grow in the pres-ence of ampicillin.


303EDVO-Kit #



Instru

ctor’s G

uid

e

Idealized Schematic of Results

The size of the protein is about 40,000 for GFP.

First Student Group

Lane 1 20 µl of standard protein markers (boiled for 5 min)Lane 2 20 µl of GFP native #1 (not boiled)Lane 3 20 µl of GFP denatured #1 (boiled for 5 min)Lane 4 20 µl of GFP native #2 (not boiled)Lane 5 20 µl of GFP denatured #2 (boiled for 5 min) Second Student Group

Lane 6 20 µl of standard protein markers (boiled for 5 min)Lane 7 20 µl of GFP native #1 (not boiled)Lane 8 20 µl of GFP denatured #1 (boiled for 5 min)Lane 9 20 µl of GFP native #2 (not boiled)Lane 10 20 µl of GFP denatured #2 (boiled for 5 min)

When proteins samples are boiled for 5 minutes in the presence of SDS and 2-mercaptoethanol, proteins lose their tertiary structure and are denatured. In the absence of these denaturing re-agents, complete denaturation is not achieved and local native structures in proteins can be maintained.

94,000 Da

67,000 Da38,000 Da

30,000 Da

20,000 Da14,000 Da

1 2 3 4 5 6 7 8 9 10

( - )

( + )


42



EDVO-Kit #In

stru

cto

r’s

Gu

ide

Study Questions and Answers

1. Exogenous DNA does not passively enter E. coli cells that are not com-petent. What treatment do cells require to be competent?

E. coli can be artifi cially induced to enter competency when they are treated with the chloride salts of the metal cations calcium, magnesium and rubidium. In addition, sudden cycles of heat and cold help to bring about competency. The metal ions and temperature changes affect the structure and permeability of the cell wall and membrane so that DNA molecules can pass through.

2. Why did the recovery broth used in this experiment not contain ampicil-lin?

The recovery broth did not contain ampicillin in order to give the cells a chance to repair themselves and to express their newly acquired genes without an immediate challenge.

3. What evidence do you have that transformation was successful?

A successful transformation will show colonies on the plate labeled (+) DNA/(+) Amp and should fl uoresce under long U.V. light.

4. What is the purpose of the control plates? Explain the difference be-tween each and why it is necessary to run each.

Positive and negative controls are designed to help interpret experi-ments by confi rming assumptions and eliminate artifactual results See experiment result and analysis section for details.

Control 1 - Untransformed cells grown on bacterial medium (-) DNA/ (-) Amp without ampicillin. The negative sign indicates cells are not

transformed (do not contain plasmid DNA). Results indicate robust growth to confi rm the viability of host bacteria.

Control 2 - Untransformed cells grown on bacterial medium (-) DNA/(+) Amp with ampicillin. Again, the negative sign indicates cells are not transformed (do not contain plasmid DNA). There is no visible bacterial growth since host cells (not transformed) are not resistant to ampicillin.

Control 3 - Host cells transformed with pGFP plasmid DNA. Since only a

small number are actually transformed, the cell mixture contains both transformed and untransformed cells. When the cell mixture is grown in the absence of ampicillin on (+) DNA/(-) Amp plates, transformed and


303EDVO-Kit #



Instru

ctor’s G

uid

e

untransformed cells will grow. The positive sign indicates cells are trans-formed with plasmid DNA.

In the actual transformation experiment, host cells are transformed with pGFP plasmid DNA and are grown on (+) DNA/(+) Amp. In a successful experiment, only transformed cells will selectively grown and express GFP. Transformed cells that express GFP will glow under long U.V. light.

5. What is the source of the fl uorescence?

The source of fl uorescence comes from the green fl uorescent protein (GFP) encoded by the plasmid.

6. Why is the molecular sieving matrix swelled prior to packing the col-umn?

When the matrix is swelled in an aqueous buffer it will form spheres which provide the sieving property for the separation of biomolecules.

7. What is the basis of molecular sieve chromatography?

Molecular sieve matrices are based on the separation of biomolecules based on size and shape. Assuming such molecules are globular, then size is the major parameter for the separation. If the biomolecules are too large to penetrate the pores in the molecular sieve, the large mol-ecules will go around the beads and will elute in the exclusion buffer with no separation. By contrast molecules that penetrate the Sephadex beads will be fractionated based on size with large to small molecules eluting sequentially.

Study Questions and Answers

Stability

Section V - Reactivity Data Unstable

Section VI - Health Hazard Data

Incompatibility

Conditions to Avoid

Route(s) of Entry: Inhalation? Ingestion? Skin?

Other

Stable

Hazardous Polymerization

May Occur Conditions to Avoid

Will Not Occur

Health Hazards (Acute and Chronic)

Carcinogenicity: NTP? OSHA Regulation? IARC Monographs?

Signs and Symptoms of Exposure

Medical Conditions Generally Aggravated by Exposure

Emergency First Aid Procedures

Section VII - Precautions for Safe Handling and Use Steps to be Taken in case Material is Released for Spilled

Waste Disposal Method

Precautions to be Taken in Handling and Storing

Other Precautions

Section VIII - Control Measures

Ventilation Local Exhaust Special

Mechanical (General)

Respiratory Protection (Specify Type)

Protective Gloves

Other Protective Clothing or Equipment

Work/Hygienic Practices

Eye Protection

Hazardous Decomposition or Byproducts

X Incompatibles

Strong oxidizers Toxic oxides of carbon, nitrogen and sulfur

X Incompaticles

Yes Yes Yes

Sensitizers may result in allergic reaction

No data

Repeated exposure may result in sensitization and possible anaphylactic shock.

No data

Ingestion: Allergic symptoms.

Eyes/Skin: Flush with water Inhalation: Move to fresh air

Wear suitable protective clothing. Sweep up

and place in suitable container for later disposal. Do not flush spilled material down sink.

Observe all federal, state, and local regulations Keep away from incompatible substances None

Yes None No None

Yes Splash or dust proof

Eye wash Wear protective clothing and equipment to prevent contact.

EDVOTEK®

Material Safety Data Sheet May be used to comply with OSHA's Hazard Communication

Standard. 29 CFR 1910.1200 Standard must be consulted for specific requirements.

IDENTITY (As Used on Label and List) Note: Blank spaces are not permitted. If any item is not applicable, or no information is available, the space must be marked to indicate that.

Section I Manufacturer's Name

Section II - Hazardous Ingredients/Identify Information

Emergency Telephone Number

Telephone Number for information

Date Prepared

Signature of Preparer (optional)

Address (Number, Street, City, State, Zip Code)

EDVOTEK, Inc.

Hazardous Components [Specific Chemical Identity; Common Name(s)] OSHA PEL ACGIH TLV

Other Limits Recommended % (Optional)

Boiling Point

Section III - Physical/Chemical Characteristics

Unusual Fire and Explosion Hazards

Special Fire Fighting Procedures

Vapor Pressure (mm Hg.)

Vapor Density (AIR = 1)

Solubility in Water

Appearance and Odor

Section IV - Physical/Chemical Characteristics Flash Point (Method Used)

Extinguishing Media

Flammable Limits UEL LEL

Melting Point

Evaporation Rate (Butyl Acetate = 1)

Specific Gravity (H 0 = 1) 2

Ampicillin

Ampicillin CAS# 7177-48-2 No data

No data No data No data


Slightly soluble Odorless, white crystaline powder

No data

N.D. = No data

N.D. N.D.

Dry chemical, carbon dioxide, water spray or regular foam Move container from fire area if possible. Do not scatter spilled material with water streams.

Avoid breathing vapors.

1121 5th Street NWWashington DC 20001

202.370.1500

202.370.1500

03-07-12

Material Safety Data SheetMay be used to comply with OSHA's Hazard Communication

Standard. 29 CFR 1910.1200 Standard must be consulted forspecific requirements.


Section IManufacturer's Name




Date Prepared



EDVOTEK, Inc.




202.370.1500

202.370.1500

Boiling Point






Solubility in Water

Appearance and Odor

Section IV - Physical/Chemical CharacteristicsFlash Point (Method Used)

Extinguishing Media

Flammable Limits UELLEL

Melting Point

Evaporation Rate(Butyl Acetate = 1)


EDVOTEK®

Column Elution Buffer

This product contains no hazardous materials as defined by the OSHA HazardCommunication Standard.

No data

No data

No data

No data

N/A

No data

Soluble

Clear liquid, no odor

No data

N.D. = No data

N.D. N.D.

Carbon dioxide, water spray

Use agents suitable for type of surrounding

May produce toxic gases

03-07-12

Stability

Section V - Reactivity DataUnstable


Incompatibility

Conditions to Avoid

Route(s) of Entry: Inhalation? Ingestion?Skin?

Other

Stable



Will Not Occur


Carcinogenicity: NTP? OSHA Regulation?IARC Monographs?




Section VII - Precautions for Safe Handling and UseSteps to be Taken in case Material is Released for Spilled



Other Precautions





Protective Gloves



Eye Protection


X None

copper, iron, silver salts, hydrogen peroxide, phenol, picric acid, formaldehydeether alcohol, nitrogen oxide, strong bases, oxidizing agents

X None

Yes Yes Yes

Toxicity has not been quantified. Sensitivity reactions may occur by skin penetration including anaphylactic shock

None identified

May cause irritation to skin/eye, mucous membranes and upper respiratory tract

Respiratory conditions

Treat symptomatically and supportively

Mop up with absorbant material. Dispose of properly

Mix with vermiculite and dry caustic, wrap in paper and burn in a chemicalincinerator equipped with afterburner and scrubber. Ignite in presence of sodium carbonate and slaked lime

Wear Protective gear to avoid skin/eye contact.

Yes None

No Process Encl. Vent. Sys.

Yes Safety goggles

Protection to avoid skin contact

Avoid skin/eye contact and inhalation

Material Safety Data Sheet May be used to comply with OSHA's Hazard Communication

Standard. 29 CFR 1910.1200 Standard must be consulted for specific requirements.


Section I Manufacturer's Name




Date Prepared



EDVOTEK, Inc.



Boiling Point






Solubility in Water

Appearance and Odor

Section IV - Physical/Chemical Characteristics Flash Point (Method Used)

Extinguishing Media

Flammable Limits UEL LEL

Melting Point

Evaporation Rate (Butyl Acetate = 1)


IPTG

Not applicable

None None None

Unknown 109-110C None

Moderate White crystals/slight odor thiophenol

Unknown Water, carbon dioxide, or dry chemical None None


202.370.1500

202.370.1500

03-07-12

Stability

Section V - Reactivity Data Unstable


Incompatibility

Conditions to Avoid

Route(s) of Entry: Inhalation? Ingestion? Skin?

Other

Stable



Will Not Occur


Carcinogenicity: NTP? OSHA Regulation? IARC Monographs?




Section VII - Precautions for Safe Handling and Use Steps to be Taken in case Material is Released for Spilled



Other Precautions





Protective Gloves



Eye Protection


X None Strong oxdizing agents Carbon dioxide and sulfur dioxide

X

Toxcity has not been studied

External: flush with water Internal: Induce vomiting, consult physician

Lab apron Avoid dust or contact with skin

Yes Not studied Not studied

Unknown No data No data No data

Unknown: avoid dust Unknown

Cover and sweep up with inert carrier Dissolve in a combustible solvent and burn in a chemical incinerator with afterburner and scrubber, or sweep up and return inoriginal container.

Avoid dust store cool Information CAS #367-93-1

Filter mask

Yes None Yes None

Rubber or vinyl Face mask or goggles

EDVOTEK®

Material Safety Data SheetMay be used to comply with OSHA's Hazard CommunicationStandard. 29 CFR 1910.1200 Standard must be consulted for

specific requirements.






Date Prepared



EDVOTEK, Inc.



Boiling Point






Solubility in Water

Appearance and Odor


Extinguishing Media


Melting Point



EDVOTEK®

Protein Denaturing Solution

Lauryl Sulfate, Sodium No data No data No data No dataC12H26O4SCAS # 151-21-3

No data

No data

No data

No data

No data

No data

Soluble

Blue liquid


Water spray, Carbon dioxide, dry chemical powder, alcohol or polymer foam

Wear SCBA and protective clothing to prevent contact with skin and eyes.

May emit toxic fumes.

Stability



Incompatibility

Conditions to Avoid


Other

Stable



Will Not Occur









Other Precautions





Protective Gloves



Eye Protection


X

Strong oxidizing agents

Carbon Monoxide, Carbon Dioxide, Sulphur oxides

X

Rubber boots

Avoid prolonged or repeated exposure

None

None

Yes Yes YesIrritating to eyes, skin, mucous membranes and upper respiratory tract.

Chronic exposure may cause lung damage or pulmonary sensitization resulting in hyperactive airway dysfunction.

Not Known No data No data No data

Respiratory tract: Burning sensation, coughing, wheezing, laryngitis, shortness of breath, headache, nausea

No data

Flush skin/eyes with large amounts of water. If inhaled, remove to fresh air.Ingestion: Large amounts of water or milk. Do not induce vomiting.

Evacuate area. Wear SCBA apparatus, rubber boots and rubber gloves. Mop up w/absorptive material and burn in chemical incinerator equipped w/ afterburner & scrubber.

Observe all federal, state, and local laws.

Wear protective gear. Avoid contact/inhalation.

Strong sensitizer

NIOSH/MSHA approved respirator

No Chemical fume hood

No None

Rubber Splash proof goggles


202.370.1500

202.370.1500

03-07-12








Date Prepared



EDVOTEK, Inc.



Boiling Point






Solubility in Water

Appearance and Odor


Extinguishing Media


Melting Point



Protein InstaStain

Methanol (Methyl Alcohol) 200ppm 200ppm No data 90%-100%CH3OH

65°C

96mmHg

1.11

.79

N/A

4.6

Complete (100%)

chemical bound to paper, no odor

(closed cup) 12°C 6.0% 36%

Use alcohol foam, dry chemical or carbon dioxide. (Water may be ineffective)

Wear SCBA with full facepiece operated in positive pressure mode. Move containers from firearea

Vapors may flow along surfaces to distant ignition sources.

Close containers exposed to heat may explode. Contact w/ strong oxidizers may cause fire.

Stability



Incompatibility

Conditions to Avoid


Other

Stable



Will Not Occur









Other Precautions





Protective Gloves



Eye Protection


X None


Carbon monoxide, Carbon dioxide, Sulfur oxides

X None

Rubber boots

Avoid prolonged or repeated exposure

Yes Yes YesIrritating to eyes, skin, mucous membranes and upper respiratory tract.

Chronic exposure may cause lung damage or pulmonary sensitization


Respiratory tract: burning sensation. Coughing, wheezing, laryngitis, shortness of breath, headache

No data

Flush skin/eyes w/ large amounts of water. If inhaled, remove to fresh air. Ingestion: give large amounts of water or milk. Do not induce vomiting.

Evacuate area. Wear SCBA, rubber boots and rubber gloves. Mop up w/ absorptive material and burn in chemical incinerato equipped w/ an afterburner and scrubber.

Observe all federal, state, and local laws.

Wear protective gear. Avoid contact/inhalation.

Strong sensitizer

NIOSH/MSHA approved respirator

No Chem fume hood

No None

Rubber Splash-proof goggles

EDVOTEK®


202.370.1500

202.370.1500

03-07-12








Date Prepared



EDVOTEK, Inc.



Boiling Point






Solubility in Water

Appearance and Odor


Extinguishing Media


Melting Point



Sephadex

Data not availableCAS# 9050-94-6

No data

No data

No data

No data

No data

No data

No data

White powder


Water spray, Carbon dioxide, dry chemical powder, foam

Wear SCBA and protective clothing to prevent contact with skin and eyes.

Emits toxic fumes under fire conditions

Stability



Incompatibility

Conditions to Avoid


Other

Stable



Will Not Occur









Other Precautions





Protective Gloves



Eye Protection


X

No data available

X

None

Do not ingest. Avoid contact with skin/eyes.

None

Yes Yes YesIrritating

Yes Yes Yes

No data available

No data

Skin/eye contact: Flush eyes with copious amounts of water Inhalation: Remove to fresh air Ingestion: Wash out mouth with water

Sweep up, place in a bag and hold for waste disposal

Normal solid waste disposal

None

None

Chemical cartridge respirator with full face mask

Yes

Yes Yes

Yes Yes

EDVOTEK®


202.370.1500

202.370.1500

03-07-12








Date Prepared



EDVOTEK, Inc.



Boiling Point






Solubility in Water

Appearance and Odor


Extinguishing Media


Melting Point



Tris-Glycine SDS Buffer (10X)

Tris CAS# 77-86-1 3%Glycine CAS# 56-40-6 14%Sodium dodecyl sulfate CAS# 151-21-3 1%

No data

No data

No data

No data

No data

No data

Soluble

Clear, no odor


Water spray, carbon dioxide, dry chemical powder or appropriate foam

Wear SCBA and protective clothing

May emit toxic fumes

Stability



Incompatibility

Conditions to Avoid


Other

Stable



Will Not Occur









Other Precautions





Protective Gloves



Eye Protection


X Strong oxidizing agents, strong acids


Carbon monoxide, carbon dioxide, sulfur oxides, sodium oxides

X

Yes Yes Yes

May cause irritation to eyes, skin, and mucous membranes.

No data No data No data No data

Irritation

Unknown

Skin/eye contact: flush w/ water. Inhalation: remove to fresh air.Ingestion: Seek medical attention

Wear protective clothing. Avoid contact. Mop up with absorbant material and dispose of properly.

Follow all state, federal, and local regulations.

Avoid contact, keep away from heat.

None

NoYes

NoneNone

Chem resistant Safety goggles

Lab coat, coveralls

Prevent contact

EDVOTEK®


202.370.1500

202.370.1500

03-07-12

EDVO-Kit # Exploring Biotechnology with the Green Fluorescent … · Biotechnology with the Green...

Documents

Transcript of EDVO-Kit # Exploring Biotechnology with the Green Fluorescent … · Biotechnology with the Green...