R e i s e d a d n U p d a t e d - EDVOTEK · The Biotechnology Education Company® ® The Biotechnology Education Company ® • 1-800-EDVOTEK • U p d a t e d R e v i s e d a d

The Biotechnology Education Company®

®

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

Updated

Revised

and

EVT 091102K

EDVO-Kit #

201Transformation of E. coli with pBR322

Storage: See Page 3 for specific storage instructions

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.

ExpERimEnT OBjEcTiVE:

The objective of this experiment is to develop an understanding of bacte-rial transformation by plasmid DNA. This experiment enables the students to observe an acquired phenotypic trait exhibited by transformed bacterial cells.

No IPTG used in this experiment.

The Biotechnology Education Company ® • 1-800-EDVOTEK • www.edvotek.comEVT 091102K

Transformation of E. coli with pBR322EDVO-Kit # 2012

Table of contents

Page

Experiment Components 4

Experiment Requirements 5

Background Information 6

Experiment Procedures

Experiment Overview 8

Laboratory Safety 9

Experimental Procedures

Protocol A (Conventional) 10

Protocol B (AP Biology) 15

Experiment Results and Analysis

Experiment Questions 20

Determination of Transformation Efficiency 22

Study Questions 23

Instructor's Guidelines

Notes to the Instructor 25

Pre-Lab Preparations

Pour Agar Plates 27

Protocol A 30

Protocol B 32

Protocol Hint and Optional Activity 34


Experiment Questions 34

Idealized Schematic of Results 36

Study Questions and Answers 37

Material Safety Data Sheets 38

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

24-hour FAX: (301) 340-0582 • email: [email protected] 091102K

EDVO-Kit # 201Transformation of E. coli with pBR322 3

After choosing a protocol option, perform all pre-lab and experimental procedures consistently in accordance with instructions for the chosen protocol.

This transformation experiment can be conducted by two different methods.

Protocol A: Conventional Method Using EDVOTEK LyphoCells™

Protocol B: Alternate Method (1997) for Advanced Placement Biology, Lab 6A

important READ mE!

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

There are subtle differ-ences between the two protocols provided with this experiment. The major difference be-tween the two options are illustrated in the figure at right.

ResuspendedFreeze-driedE. coli cells

Protocol AConventional

Method

Protocol BAP Biology

Lab 6A

Incubate vial of cells

overnight at 37°C

Add CompetencyInduction Solvent

and Incubate on icefor 30 minutes

Aliquot for each group

0.7 ml Cell Suspension

Plate cells and growovernight at 37°C

Students begin Experiment Protocol A

Students begin Experiment Protocol B

ce

lls

Students transfer

300 µl Cells

Co

ntro

lBu

ffer

pBR

322

DN

A

Inst

ruc

tor’

Pre

-la

b P

rep

Instructo

r’ Pre-la

b Pre

p

Studentstransfer 2-3

large colonies

250 µlCaCl2

+DN

A

-DN

A



Storage: Store components A-G in the refrigerator.

A Transformation LyphoCells™ (DO NOT FREEZE) B Supercoiled pBR322 DNA C Control Buffer (no DNA) (not used in Protocol B) D Ampicillin E Cell reconstitution medium F Solvent for induction of competency (not used in Protocol B) G CaCl2 (not used in Protocol A)

Storage: Store components listed below at Room temperature

• BottleReadyPour™LuriaBrothAgar,sterile (also referred to as ReadyPour medium)

• BottleLuriaBrothMediumforRecovery,sterile (also referred to as Luria Recovery Broth)

• Petri plates, small

• Petri plates, large

• Plastic microtipped transfer pipets

• Wrapped 10 ml pipet (sterile)

• Toothpicks (sterile)

• inoculating loops (sterile)

• Microtest tubes with attached lids

Experiment components

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

None of the experiment components are derived from human sources.

Component Quantities:

Experiment # 201 is designed for 10 groups (2-4 students per group).

impORTAnT READ mE!

in this experiment, antibiotics are used for the selection of transformed bac-teria. Students who have or suspect to have allergies to penicilllin, ampicil-lin or tetracycine should not participate in this experiment.




Technical ServiceDepartment

FAX: (301) 340-0582web: www.edvotek.comemail: [email protected]

•AutomaticMicropipet(5-50µl)andtips

•TwoWaterbaths*(37°Cand42°C)

•Thermometer

•IncubationOven

•Pipetpumpsorbulbs

•Markingpens

•Bunsenburner,hotplateormicrowaveoven

•Hotgloves

•Ice

* If a second water bath is not available, water can be heated to 42°C in a beaker. The cells will require this temperature for only a few minutes. Alternatively, 42°C water can be put in a small styrofoam container with a cover. The temperature needs to be held at 42°C.

Requirements

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

Visit our web site for information about EDVOTEK's complete

line of experiments for biotechnology and biology education.

Online Ordering now available

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


Duplication of this document, in conjunction with use of accompanying reagents, is permitted for classroom/labora-tory use only. This document, or any part, may not be reproduced or distributed for any other purpose without the written consent of EDVOTEK, Inc. Copyright © 1987, 88, 89, 90, 93, 94, 95, 97, 98, 99, 2005, 2009 EDVOTEK, Inc., all rights reserved EVT 091102K

Transformation of E. coli with pBR322EDVO-Kit # 2016B

ackg

rou

nd

info

rmat

ion

Bacterial Transformation

Number oftransformants

per µg =

100 transformants

0.01 µg

Specific example:

X

final 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 Efficiency Calculation

Bacterial transformation is of central importance in molecular biology. It allows for the propagation of recombinant DNA molecules that are con-structed in vitro or found in nature. Transformation is also of historical importance since it led to the discovery by Avery, in the late 1940’s, that DNA was the genetic material.

The transformation process involves the uptake of exogenous DNA by the cell which results in a newly acquired genetic trait that is stable and heritable. Bacteri-al cells must be in a particular physiologi-cal state before they can be transformed. This state is referred to as competency. Competency can occur naturally in cer-tain species of Haemophilus and Bacillus when the levels of nutrients and oxy-gen are low. Competent Haemophilus express a membrane associated trans-port complex which binds and transfers certain DNA molecules from the medium into the cell where they are incorporated and their genes are expressed. In nature, the source of external DNA is from other cells that have died and lysed.

Much of the current research and experimentation in molecular biology involves the transformation of E. coli which does not enter a stage of natural competency. E. coli can be artificially induced to enter compe-tency upon treatment with the chloride salts of cations, such as calcium, magnesium and rubidium. Sudden cycles of heat and cold help to bring about competency. Metal ions and temperature changes affect the structure and permeability of the cell wall so that DNA molecules can pass through. The reasons why this occurs are still unknown. Compe-tent E. coli cells are fragile and must be treated with care.

The amount of cells transformed per 1 microgram of DNA is called the transformation efficiency. In practice, much smaller amounts of DNA are used (5 to 100 nanograms) since higher concentrations of DNA inhibit the transformation process. Typically, 10 nanograms (0.01 microgram) of DNA is used to transform cells that are in a final volume of 1 ml. After transformation, the cell suspension is plated on agar medium in the presence of the antibiotic (coded by the transforming plasmid). This allows the selection of transformed cells since the presence of antibiot-ics will inhibit the growth of cells that had not acquired the antibiotic resistance gene.

After the incubation of plates with the transformed cells, colonies will appear on the plate. Keeping in mind that each colony originally



EDVO-Kit # 201Transformation of E. coli with pBR322 7B

ackgro

un

d in

form

ation

Bacterial Transformation

grew from one transformed cell, the transformation efficiency can be calculated. For example, if 100 colonies are present, the transforma-tion efficiency can be calculated as outlined in Figure 1. In research laboratories, bacterial transformation efficiencies generally range from 1 x 106 to 1 x 108 cells per microgram of DNA. There are procedures which can produce cells having transformation efficiencies approaching 1010. Transformation is never 100%; in a typical experiment, 1 in 10,000 cells successfully incorporate the transforming plasmid. However, there is such a large number of bacterial host cells in a sample (typically 1 x 109) and only a small fraction of cells need to be transformed to obtain colonies on a plate. Transformation efficiency can be demonstrated by plating equal volumes (0.1 ml) of recovered cells on selective (contain-ing antibiotic) and nonselective bacterial growth agar medium. The nonselective medium, which does not contain the antibiotic, will have a large number of cells known as a “lawn”. Bacterial agar plates will be covered heavily with untransformed cells, forming a "lawn" in contrast to individual colonies.

Plasmid DNAs are extrachromosomal, double-stranded circular molecules that are found in various strains of bacteria. Many plasmids contain genes that code for antibiotic resistance. E. coli plasmid pBR322 consists of 4,362 base pairs and contains antibiotic resistance genes for ampicillin (Amp) and tetracycline (Tet). Ampicillin is a derivative of penicillin and inhibits bacterial growth by interfering with the synthesis of cell walls. The product of the ampicillin resistance gene is beta-lactamase. This enzyme is secreted by transformed bacterial cells and destroys the ampicillin in the surrounding agar medium. Subsequently, cells that were not transformed are able to undergo limited growth in the zones that have been cleared of ampicil-lin. Colonies consisting of untransformed cells typically form small colonies that are called satellites since they only appear around the larger transformed colonies. Larger plating volumes of cells and lon-ger incubation times increase the amount of satellite colonies.

In bacterial cells, plasmids are present primarily in supercoiled forms. In that conformation, the two strands of DNA are condensed entangled structures compared to the relaxed (non-supercoiled) DNA (Figure 2). Competent E. coli cells are sensitive to the conformation of DNA and are selective to the supercoiled plasmid form during transformation.

Supercoiled Relaxed

Figure 2: Supercoiled and circular forms of plasmid DNAs



Transformation of E. coli with pBR322EDVO-Kit # 2018Th

e Ex

per

imen

t

Experiment Overview

BEfORE yOu START ThE ExpERimEnT

1. Read all instructions before starting the experiment.

2. Write a hypothesis that reflects the experiment and predict experi-mental outcomes.

ExpERimEnT OBjEcTiVE:

The objective of this experiment is to develop an understanding of bacterial transformation by plasmid DNA. This experiment enables the students to observe an acquired phenotypic trait exhibited by trans-formed bacterial cells.

BRiEf DEScRipTiOn Of ExpERimEnT:

In this experiment, you will transform a strain of competent E. coli which has no antibiotic resistance with supercoiled pBR322 DNA. The cells will be selected for the presence of plasmid by plating them on agar medium containing ampicillin. The transformation efficiency will then be estimated.

This transformation experiment can be conducted by two different methods.

Protocol A: Conventional Method Using EDVOTEK LyphoCells™

Protocol B: Alternate Method (1997) for Advanced Placement Biology, Lab 6A

Two protocols are provided with this experiment. There are subtle differences throughout the two experiment protocols. Please make sure you are following the appropriate protocol as determined by your instructor.



EDVO-Kit # 201Transformation of E. coli with pBR322 9Th

e Experim

ent

Laboratory Safety

important READ mE!

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

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 patho-genic. 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 labo-ratory disinfectant.

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

• Autoclaveat121°Cfor20minutes. 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.

• Soakin10%bleachsolution. Immerse petri plates, open tubes and other contaminated mate-

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

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




e Ex

per

imen

t

protocol A - conventional method using EDVOTEK Lyphocells™

Incubate on ice for 10 minutes

Incubate at 42°Cfor 90 seconds

Place on icefor 1 minute

Add 0.75 ml recovery broth

Incubate in 37°Cwater bath for 30 minutes

Streak 0.25 ml of cell suspension on plates

Incubateovernight in 37°Cincubation oven

Incubateovernight in 37°Cincubation oven

NO AMPControl

PLUS AMPControl

NO AMPpBR322 DNA

PLUS AMPpBR322 DNA

White Colonies No Colonies White Colonies White Colonies

25 µl pBR322 DNA

0.7 mlCells

25 µl Control Buffer

Control Buffer

0.25 mlCells

pBR322 DNA

0.25 mlCells

Plates contaning antibiotic have a stripe on the side of the plate.

NO AMPControl

PLUS AMPControl

NO AMPpBR322 DNA

PLUS AMPpBR322 DNA

For optimal results, store covered plates in the up-right position after streaking to allow the cell suspen-sion to be absorbed by the agar. After approximately 20 minutes, invert the plates for overnight incubation at 37°C.

TRAnSfORmATiOn ExpERimEnT fLOw chART




e Experim

ent


SETTinG up ThE TRAnSfORmATiOn AnD cOnTROL ExpERimEnT

1. Put your initials or group number on the tubeslabeled“pBR322DNA”(contains25µlof plasmid DNA) and “Control Buffer” (con-tains25µlofbuffer).Placethembackonice.

2. Set up the Control:

• Usingasteriletransferpipet,transfer0.25ml(250µl)ofthecellsuspension to the tube “Control Buffer”.

• Carefullyplacethepipetbackintothe wrapper.

• Capthetube;mixbytapping.Putthe tube back on ice.

3. Set up the transformation:

• UsingthesamepipetfromStep2, transfer0.25ml(250µl)of the cell suspension to the tube “pBR322 DNA”.

• Capthetube;mixbytapping.Putthetubebackonice.

4. Incubate the cells prepared in steps 1 - 3 on ice for 10 minutes.

5. Place both tubes in a waterbath at 42°c for 90 seconds.

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

6. Return both tubes to incubate on ice for for 1 minute.

Co

ntro

lB

uffe

r

pBR

322

DN

A-

DN

A

+ D

NA

Guide for Sterile Calibrated

Transfer Pipet

1 ml

0.5 ml

0.25 ml

~ 0.06 ml

< 0.02 ml

0.75 ml

Con

trol

Buf

fer

pBR

322

DN

A

- D

NA

+ D

NA

42°c

- D

NA

+ D

NA




e Ex

per

imen

t

- D

NA

+ D

NA

37°c

7. Add 0.75 ml of the Recovery Broth to the tube “Control Buffer”.

Add the recovery broth with a sterile 1 ml pipet. Avoid touching the cells with the pipet.

8. Add 0.75 ml of the Recovery Broth to the tube “pBR322 DNA”.

pBR

322

DN

A

Co

ntro

lB

uffe

r

Co

ntro

lB

uffe

r

pBR

322

DN

A


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

• Labeloneunstripedplate: NOAMPControl • Labeloneunstripedplate: NOAMPpBR322DNA

• Labelonestripedplate: PLUSAMPControl

• Labelonestripedplate: PLUSAMPpBR322DNA

• Putyourinitialsorgroupnumberonalltheplates.

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

9. Incubatetheclosedtubesina37°Cwaterbath for 30 minutes for a recovery pe-riod.

Quick Reference:

DNA and competent cells are combined in a 0.25 ml suspension. 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.




e Experim

ent

pLATinG ThE cELLS

plating cells from the tube labeled "control":

12. Use a fresh, sterile 1 ml pipet to transfer recovered cells from the tube "Control Buffer" to the middle of the following plates:

• 0.25mltotheplatelabeled NOAMPControl • 0.25ml totheplatelabeled PLUSAMPControl

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 spread-ing. Be careful to avoid gouging the loop into the agar.

Reminder:Follow proper procedures for disposal of contaminated materials.


13. Spread the cells over tne entire plate with a sterile inoculating loop.

14. Cover both control plates and allow the liquid to be absorbed.

Spread cells in one direction

Same plate -spread cells 90° to first direction

plating cells from the tube labeled "pBR322 DnA"

15. Use a fresh, sterile 1 ml pipet to transfer recovered cells from the tube "pBR322 DNA" to the middle of the following plate:

• 0.25mltotheplatelabeled NOAMPpBR322DNA • 0.25ml totheplatelabeled PLUSAMPpBR322DNA

16. Spread the cells with a sterile inoculating loop.

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




e Ex

per

imen

t

pREpARinG pLATES fOR incuBATiOn

18. Stack your group's set of plates on top of one another and tape them together.

19. Put your initials or group number on the taped set of plates.

20. Place the set of plates in a safe place designated by your instructor. The plates should be left in the upright position to allow the cell suspension to be absorbed by the agar for 15 - 20 minutes.

21. Place the plates in the inverted position (agar sideontop)ina37°Cbacterialincubationoven 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 condensa-tion on the lid, which could drip onto the culture and interfere with experimental results.

ViEwinG pLATES AfTER incuBATiOn

22. Proceed to analyzing your results.

23. After analyzing your results, follow proper procedures for disposal of contaminated materials.

protocol A - conventional method using EDVOTEK Lyphocells™G

rou

p 4


If you do not have an incu-bation oven, the plates can be left at room tempera-ture. Colonies of trans-formed cells should appear between 24 - 48 hours.




e Experim

ent

+pBR322

DNA(pAMP)

Add 0.25 mlLuria

Recovery Broth

Incubate on icefor 2 minutes

Incubate at 42°Cfor 90 seconds

Incubate on icefor 15 minutes

Streak 0.25 ml of + DNA cells

on each plate

Streak 0.25 ml of - DNA cells

on each plate

Incubate overnight in 37°Cincubation oven

Incubate overnight in 37°Cincubation oven

- D

NA

Transfer 2-3 large colonies

0.25 mlCaCl2

Prepare Source PlatesDAY BEFORE LAB

37°C for30 minutes

+ D

NA

- D

NA

+ D

NA

PLUS AMPpBR322 DNA

NO AMPpBR322 DNA

PLUS AMPControl

NO AMPControl

E.coli cells

Plates contaning antibiotic have a stripe on the side of the plate.

10 µl

PLUS AMPpBR322 DNA

NO AMPpBR322 DNA

PLUS AMPControl

NO AMPControl

protocol B - Advanced placement Biology Lab 6A

TRAnSfORmATiOn ExpERimEnT fLOw chART

* The 37°C incubation for

30 minutes (after adding the Recovery Broth) is not described in the AP Biology Student Laboratory Manual (1997 Edition D). However, EDVOTEK recommends that you perform this step to allow the cells to recov-er and begin to express the antibiotic resistance genes. The Recovery Broth does not contain antibiotic.




e Ex

per

imen

t

+

E. coli CellsSource Plate

250 µlCaCl2

pBR322

Transfer 2-4 large colonies to each tube containing

ice cold CaCl2

- D

NA

+ D

NA

10 µl

5. Suspend the cells in both tubes by tapping or vortexing.

6. Add10µlofpBR322DNAtothetube labeled “ + DNA “.

This plasmid contains the ampicillin resistance gene, often referred to as pAMP.

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

SETTinG up ThE TRAnSfORmATiOn AnD cOnTROL ExpERimEnT

1. Label one microcentrifuge tube “ + DNA “. This will be the trans-formation tube with plasmid DNA.

2. Label a second microcentrifuge tube “ - DNA “. This will be the experimental control tube without plasmid DNA.

3. Usingasterile1mlpipet,add250µl(0.25 ml) of ice cold CaCl2 solution to each tube.

4. Pick colonies from the source plate of E. coli cells to each of the test tubes labeled “ + DNA “ and “ - DNA “:

• useasteriletoothpicktotransfer2colonies(2-4mm)fromthesource plate to the test tubes.

• Betweenyourfingers,twistthetoothpickvigorouslyandupand down in the CaCl2 solution to dislodge and emulsify the cells.

+ D

NA

- D

NA


- D

NA

+ D

NA

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 resuspended in the calcium chloride solution and is not left on the toothpick or on the wall of the tube.

Contains plasmid DNA

Does not contain plasmid DNA

“+”

“-”

Reminder




e Experim

ent

+ D

NA

- D

NA


- D

NA

+ D

NA

8. Place both tubes in a waterbath at 42°c for 90 seconds for the heat shock step. This facilitates the entry of DNA in bacte-rial cells.

- D

NA

+ D

NA

42°c

9. Return both tubes im-mediately to the ice bucket and incubate for two minutes.

- D

NA

+ D

NA

- D

NA

+ D

NA

37°c * This step (#11) is not described in the

AP Biology Student Laboratory Manual (1997 Edition D). However, EDVOTEK recommends that you perform this step to allow the cells to recover and begin to express the antibiotic resistance genes. The Recovery Broth does not contain antibiotic.

10. Using a sterile pipet, add 250 µl (0.25 ml) of Recovery Broth to each tube and mix.

11. Incubate the cells for 30 minutesina37°Cwaterbathforarecovery period.*

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

• Labeloneunstripedplate: NOAMPControl • Labeloneunstripedplate: NOAMPpBR322DNA

• Labelonestripedplate: PLUSAMPControl • Labelonestripedplate: PLUSAMPpBR322DNA

• Putyourinitialsorgroupnumberonalltheplates.

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




e Ex

per

imen

t


pLATinG ThE cELLS

plating cells from the tube labeled " - DnA "

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

• 0.25mltotheplatelabeled NOAMPControl • 0.25ml totheplatelabeled PLUSAMPControl

15. Spread the cells over the entire plate with a sterile inoculating loop.

To avoid contamina-tion 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.

Spread cellsin one direction

Same plate: spread cells 90° to

first direction

16. Cover both plates and allow the liquid to be absorbed (approxi-mately 15-20 minutes).

plating cells from the tube labeled "+ DnA"

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

• 0.25mltotheplatelabeled NOAMPpBR322DNA • 0.25ml totheplatelabeled PLUSAMPpBR322DNA 18. Spread the cells with a sterile inoculating loop. 19. Cover the plate and allow the liquid to be absorbed (approximately

15-20 minutes)..

Contains plasmid DNA

Does not contain plasmid DNA

“+”

“-”

Reminder




e Experim

ent


pREpARinG pLATES fOR incuBATiOn

18. Stack your group's set of plates on top of one another and tape them together.

19. Put your initials or group number on the taped set of plates.

20. Place the set of plates in a safe place designated by your instructor. The plates should be left in the upright position to allow the cell suspension to be absorbed by the agar for 15 - 20 minutes.

21. Place the plates in the inverted position (agar sideontop)ina37°Cbacterialincubationoven 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

22. Proceed to analyzing your results.

23. After analyzing your results, follow proper procedures for disposal of contaminated materials.

Gro

up

4


If you do not have an incu-bation oven, the plates can be left at room tempera-ture. Colonies of trans-formed cells should appear between 24 - 48 hours.




e Ex

per

imen

t

LABORATORy nOTEBOOK REcORDinGS:

Address and record the following in your laboratory notebook or on a separate worksheet.

Before starting the Experiment:

• Writeahypothesisthatreflectstheexperiment. • Predictexperimentaloutcomes.

During the Experiment: • Record(draw)yourobservations,orphotographtheresults.

following the Experiment: • Formulateanexplanationfromtheresults. • Determinewhatcouldbechangedintheexperimentifthe

experiment were repeated. • Writeahypothesisthatwouldreflectthischange.

AnSwER ThESE quESTiOnS BEfORE AnALyzinG yOuR RESuLTS.

1. On which plate(s) would you expect to find bacteria most like the original non-transformed E. coli cells (Protocol B)? Explain.

2. On which plate(s) would you find only genetically transformed bac-terial cells? Explain.

3. What is the purpose of the control plates? Explain the difference between each and why it is necessary to run each.


Continued




e Experim

ent

DATA cOLLEcTiOn

1. Observe the results you obtained on your transformation and con-trol plates.

Transformation Plate: + DNA

• NOAMPpBR322DNA=LBmediumwithcellstransformedwith plasmid

• PLUSAMPpBR322DNA=LBmediumcontainingampicillinwith cells transformed with plasmid

Control Plates: - DNA

• NOAMPControl=LBmediumwithcellsNOTtransformedwithplasmid

• PLUSAMPControl=LBmediumcontainingampicillinwithcells

NOT transformed with plasmid

2. Draw and describe what you observe. For each of the plates, record the following:

• Howmuchbacterialgrowthdoyouobserve?Determineacount.

• Whatcolorarethebacteria? • Whydodifferentmembersofyourclasshavedifferenttransfor-

mation efficiency values? • Ifyoudidnotgetanyresults,whatfactorscouldbeattributed

to this fact?





e Ex

per

imen

t

DETERminATiOn Of TRAnSfORmATiOn EfficiEncy

Transformation efficiency is a quantitative determination of how many cellsweretransformedper1µgofplasmidDNA.Inessence,itisanindi-cator of how well the transformation experiment worked.

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

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

PLUS AMP pBR322 DNA

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

2. Determine the transformation efficiency using the formula:

x =

final 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

0.025 µg

6400(6.4 x 103)

transformants per µg

x 1.0 ml

0.25 ml=

Quick Reference for Expt. 201 - Protocol A:

25 ng (0.025 µg) of DNA is used.

The final volume at recovery is 1.0 ml. The volume plated is 0.25 ml.

Quick Reference for Expt. 201 - Protocol B:

10 ng (0.01 µg) of DNA is used.

The final volume at recovery is 0.50 ml. The volume plated is 0.25 ml.




e Experim

ent

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

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

2. Why did the recovery broth not contain ampicillin?

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

4. What are some reasons why transformation may not be successful?

Study questions






instructor’s Guide

impORTAnT READ mE!

Transformation experiments contain antibiotics which are used for the selection of transformed bacteria. Students who have allergies to an-tibiotics such as penicilllin, 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 implemen-tation of this experiment with your students.

The guidelines that are presented in this manual are based on ten labo-ratory groups consisting of two, or up to four students. The following are implementation guidelines, which can be adapted to fit your specific set of circumstances. If you do not find 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 knowledge-able technical staff.

Day 1: (prior to the Lab) • Prepareagarplates• PrepareE. coli Cells (overnight incuba-

tion). • DispensetheDNAandcontrolbuffer

Day 2: (Day of Lab Experiment)

• Equilibratewaterbathsat37°Cand42°C;incubationovenat37°C

• Studentstransformcellsandplateforovernight incubation.

Day 3: (Day after Lab Experiment)

• Studentsobservetransformantsandcontrols

• Studentscalculatetransformationef-ficiency

• Followcleanupanddisposalproceduresas outlined in the Laboratory Safety sec-tion.

Technical ServiceDepartment

FAX: (301) 340-0582web: www.edvotek.comemail: [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

notes to the instructor:

Visit our web site for information about

EDVOTEK's complete line of experiments for

biotechnology and biology education.

Online Ordering now available



Transformation of E. coli with pBR322EDVO-Kit # 20126in

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 scientific inquiry, learn new techniques using several types of biotech-nology equipment, and will learn standard procedures used in transfor-mation. Analysis of the experiments will provide students the means to transform an abstract concept into a concrete explanation.

AppROximATE TimE REquiREmEnTS

if performing protocol A:

• ReconstituteandincubatetheLyphoCells™at37°Cfor16to24hours before the laboratory (overnight incubation).

• Competentcellsmustbedispensedjustpriortothelabexperiment.If tubes are already labeled, dispensing will require approximately 15 minutes.

if performing protocol B:

• ReconstitutetheLyphoCells™,plateforindividualcolonies,andincubateat37°Cfor16to24hoursbeforethelaboratory(overnightincubation).

LABORATORynOTEBOOKS

It is highly recommended that students maintain a laboratory notebook to formulate hypotheses and to record experimental procedures and results.

• EDVOTEKCat.#1401,Laboratory DataBook is recommended.

• Guidelinesforkeepinga laboratory notebook is available at the EDVOTEK web site.

for BOTh protocols A and B:

1. The agar plates can be prepared several days in advance and stored inverted (agar side on top) in the refrigerator. Prepara-tion requires approximately 1 hour.

2. Dispensing the DNA and reagents requires approximately 30 minutes. This can be done the day before the lab and stored in the refrigerator.

3. Allow ample time for the equilibration of the water baths at 37°Cand42°Candabacterialincubationovenat37°Contheday of the experiment.

4. In the experiment, each group will perform the transformation experiment and plate four sets of bacterial cells. These proce-dures require approximately 50 minutes.

5. Overnight incubation of plates is approximately 15-20 hours at 37°C.Colonieswillalsoappearbetween24-48hoursatroomtemperature.



EDVO-Kit # 201Transformation of E. coli with pBR322 27in

structo

r’s Gu

ide

pre-Lab preparations

pOuR AGAR pLATES (prior to the Lab experiment)

• Foroptimalresults,prepareplatestwodayspriortoplatingandsetaside the plates inverted at room temperature.

• Iftheyarepouredmorethantwodaysbeforeuse,theyshouldbestored 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. Equilibrateawaterbathat60°Cforstep5below.

2. Loosen, but do not remove, the cap on the 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 bottle to break up the solid agar into chunks

4. Heat the bottle of ReadyPour medium by one of the methods out-lined below. When completely melted, the amber-colored solution should appear free of small particles.

A. Microwave method:

• HeatthebottleonHighfortwo30secondintervals. • Usingahotglove,swirlandheatonHighforanadditional

25 seconds, or until all the medium is dissolved. • Usingahotglove,occasionallyswirltoexpeditemelting.

B. Hot plate or burner method:

• Placethebottleinabeakerpartiallyfilledwithwater. • Heatthebeakertoboilingoverahotplateorburner. • Usingahotglove,occasionallyswirltoexpeditemelting.

5. Allow the melted ReadyPour medium to cool. Placing the bottle in a60°Cwaterbathwillallowtheagartocool,whilepreventing it from prematurely solidifying.

When the ReadyPour™ medium reaches approximately 60°C, the bottle will be warm to the touch but not burn-ing hot.

Wear Hot Gloves and Goggles during all steps involving heating.

60˚C




stru

cto

r’s

Gu

ide


Label (“Stripe”) the plates

6. Use a lab marker to "stripe" the sides of twenty (20) 60 x15 mm petri dishes. This will provide an easy method of differentiating between plates with ampicillin and plates without ampicillin.

• Openonesleeveof20platesandstacktheplatesneatly. • Startthemarkeratthebottomofthestackandmovethe

marker vertically to the top plate to "stripe" the sides of the 20 plates.

• Theseplateswillbeusedformediumwithampicillin. • Donotstripethesecondsleeveofplates.Thesewillbethe

control plates.

pour the plates (after the medium has cooled)

Note: If performing Protocol B, five (5) source plates are required. Source Plates are not required for Protocol A. Both Protcols A and B require 20 control plates and 20 Amp plates.

7. Pour 5 large E. Coli source plates (protocol B only)

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

8. Pour 20 control plates (no ampicillin, no-stripe):

Use a fresh 10 ml pipet (or the same pipet from step 7) and pipet pump to pour the 20 control plates, 5 ml each with ReadyPour me-dium without ampicillin.

• 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 flame across the surface of the medium.

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

quick Reference: pouring Agar plates




structo

r’s Gu

ide

Add reagents to me-dium which has been cooled. Hot medium will cause reagents such as ampicillin to rapidly decompose.

9. Add the entire amount of ampicillin powder to the remaining mol-ten ReadyPour medium in the bottle.

10. Recap the bottle and swirl to completely dissolve the ampicillin.

11. Pour 20 transformation plates (with ampicillin, striped plates):

Use a fresh 10 ml pipet to pour the twenty (20) striped plates, 5 ml each.

12. 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, in-verted.

If you have extra sterile petri plates on hand, use any remaining me-dium to pour additional plates for the optional activity described on page 34.



Summary of Poured PlatesProtocol A

20 control plates - small no stripe plates: 5 ml each - ReadyPour medium (no ampicillin)

20 transformation plates - small striped plates: 5 ml each - ReadyPour medium with ampicillin

Summary of Poured PlatesProtocol B

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

20 control plates - small no stripe plates: 5 ml each - ReadyPour medium (no ampicillin)

20 transformation plates - small striped plates: 5 ml each - ReadyPour medium with ampicillin




stru

cto

r’s

Gu

ide

pre-Lab preparations - protocol A

DAy BEfORE ThE ExpERimEnT

preparation of E. coli cells

1. Use a 10 ml sterile pipet to add 6 ml cell reconstitu-tion medium (E) to the vial of LyphoCells.

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

3. Incubatethevialat37°Cfor16-24hours(overnight)in an incubation oven. For optimal results, incubate LyphoCellsat37°Cfor19hours.

DAy Of ThE ExpERimEnT

preparation of E. coli cells, continued

4. Completely thaw the competency induction solvent (F) and place on ice. (If there is a white precipitate inthebottle,warmitina37°Cwa-terbath to dissolve the precipitate.)

5. Mix and resuspend the vial of incu-bated cells by inverting and gently shaking. Place the vial on ice for 10 minutes.

6. Use a 10 ml sterile pipet to add 3 ml of ice cold com-petency induction solvent (F) to the vial of cells.

The competency induction solvent is very viscous. Make sure that a portion of the solvent is not left on the walls of the pipet.

7. Mix the cells and induction solvent thoroughly by inverting the vial several times. The solution should have no dense layers, "streams" or globules (i.e. it should be a uniform suspension).

8. Keep the cells on ice for a minimum of 30 minutes.

Cells can be kept on ice for up to 3 hours.

The solvent for induction of competency contains salts to make cells competent.

Add 6 ml reconstitution media (E) toLyphoCells

Place vial ofcells on ice10 minutes

Dispense0.7 ml Cell

Suspension

Add 3 mlice cold

CompetencyInduction Solvent (F)

Incubate vial of cellsovernight at 37°C

Ce

lls

Incubate on ice30 minutes

Students begin Experiment Protocol A




structo

r’s Gu

ide

pre-Lab preparations - protocol A

Dispensing the cells (just prior to the Experiment:)

Keep cells on ice during dispensing procedures:

11. Mix the cells by inversion to obtain an even suspension.

12. Use a sterile pipet to aliquot 0.7 ml of cells to 10 ice cold tubes labeled "Cells"

13. Cap the tubes and keep them on ice.

preparation of DnA and control Buffer

14. Place the tubes of supercoiled pBR322 DNA (B) and Control Buffer (C) on ice.

Note: This control buffer is not used in Protocol B.

15. Before dispensing the DNA and control buffer, tap the tubes until all the sample is at the tapered bottom of the tube.

16. Using an automatic micropipet, dis-pense25µlofthesupercoiledpBR322DNA to each of 10 microtest tubes labeled "pBR322 DNA".

17. Cap the tubes and place them on ice.

18. Using a FRESH micropipet tip, dispense 25µlofcontrolbuffertoeachof10mi-crotest tubes labeled "Control Buffer".


Recovery Broth

Set up a classroom pipeting station, or dis-pense recovery broth (optional).

20. Dispense 1.5 ml Recovery Broth into 10 sterile tubes labeled "Recovery Broth" using a sterile pipet.

21. Cap the tubes and place them in the re-frigerator if not to be used immediately.

Each Group Requires:

1 tube pBR322 DNA 25 µl on ice1 tube Cells 0.7 ml on ice1 tube Control Buffer 25 µl on ice2 striped plates2 unstriped plates4 sterile 1 ml pipets2 sterile inoculating loops1 tube Recovery broth (optional) 1.5 ml

Summary of Reagent Preparations 20 plates NO AMP (No Stripe) 5 ml20 plates PLUS AMP (Stripe) 5 ml10 tubes pBR322 DNA 25 µl on ice 10 tubes Control Buffer 25 µl on ice10 tubes Cells 0.7 ml on ice10 tubes Recovery Broth 1.5 ml




stru

cto

r’s

Gu

ide

pre-Lab preparations - protocol B

DAy BEfORE ThE ExpERimEnT

This experiment requires preparation of isolated E.coli host transformation colonies 16-24 hours before the laboratory ex-periment, so plan accordingly.

important: Do not prepare source plates more than 24 hours before the experiment. Old source plates will compromise the success of the transformation experiment.

preparation of E. coli cells

1. Use a 10 ml sterile pipet to add 6 ml of cell reconstitution medium (E) to the vial of LyphoCells.

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

3. Incubatethevialofcellsfor30-60minutesina37°Cincuba-tion 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. Using a sterile loop, dip into the cell suspension to remove a loop full of bacterial. Plate the inoculum on one quadrant of the plate with the loop (figure top right).

5. With the same loop, streak through the cells once or twice into another clean section of the plate (figure bottom right) to obtain isolated colonies.

6. Label the plates "E. coli", invert and incubate the plates overnight(16-24hours)at37°Cinanincubationoven.

If growth results on plates is heavy (i.e. few or no isolated colonies), instruct students to touch the toothpick to a small amount of cells.

LB Agar

Incubate Source platesovernight at 37°C

Add 6 ml reconstitution media (E) to

LyphoCells vial

Incubate cells30 - 60 minutes at 37°C

Transfer 0.3 ml to each Source plate

E. coli CellsSource Plate

Students begin Experiment Protocol B

Studentstransfer 2-3

large colonies

250 µlCaCl2

+DN

A

-DN

A




structo

r’s Gu

ide

DAy Of ThE ExpERimEnT

preparation of DnA and Reagents

7. Place the tube of supercoiled pBR322 DNA (B) on ice.

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

9. Usinganautomaticmicropipet,dispense12µlofthesupercoiledpBR322 DNA to each of 10 microtest tubes labeled "pBR322 DNA".


11. Dispense 1 ml of CaCl2 (G) into microcentrifuge tubes labeled "CaCl2" for each of the 10 groups and place on ice.

Note: CaCl2 is not used in Protocol A.

pre-Lab preparations - protocol B

Summary of Reagent Preparations

5 plates E. coli cells20 plates NO AMP (No Stripe) 5 ml20 plates PLUS AMP (Stripe) 5 ml10 tubes pBR322 DNA 25 µl on ice 10 tubes Control Buffer 25 µl on ice10 tubes Recovery Broth (optional) 1.5 ml

Recovery Broth

Dispense recovery broth (optional) or set up a classroom pipeting station.

12. Dispense 1.5 ml Recovery Broth into 10 sterile tubes labeled "Recovery Broth" using a sterile pipet.

13. Cap the tubes and place them in the re-frigerator if not to be used immediately.

Each Group Requires:

• 1 tube (12 µl) pBR322 DNA (on ice) • 1 tube (1 ml) CaCl2 (on ice) • 1 plate E. coli (every 2 groups can share) • 2 empty microcentrifuge tubes • 2 toothpicks • 2 striped plates • 2 unstriped plates • 4 sterile 1 ml pipets • 2 sterile inoculating loops • 1 tube (1.5 ml) Recovery broth (optional)




stru

cto

r’s

Gu

ide

AnSwER ThESE quESTiOnS BEfORE AnALyzinG yOuR RESuLTS.

1. On which plate(s) would you expect to find bacteria most like the original non-transformed E. coli cells (protocol B)? Explain.

Bacteria on the plate labeled “NO AMP Control” would be identical to the non-transformed starter E. coli source plate because they are not transformed by plasmid. Cells were re-plated onto a NO AMP plate.

2. On which plate(s) would you find only genetically transformed bacterial cells? Explain.

Bacteria growing on the plate labeled “PLUS AMP pBR322 DNA” are transformed. Cells will express the ampicillin resistance gene and will survive on the plates which contain ampicillin.

protocol hint and Optional Activity:

Do not discard the tubes containing transformed bacteria. After plating analiquotonselectionplates,addanadditional50µlofrecoverybrothto the tubes and set them in a rack. Leave on the lab bench overnight. If for some reason, transformants do not grow on the selection plates, the remaining cells can be plated as outlined below:

1. Collect the bacterial cell pellet by centrifugation in a microcentri-fuge. If a microcentrifuge is not available, let the bacteria collect by gravity and do not disturb.

2. Removeallexcept50µlofmedium(supernatant,toplayer).

3. Resuspend the cell pellet in remaining medium.

4. Spread the entire contents of the tube on selection medium.

5. Incubatetheplateasbefore,16-24hoursina37°Cincubationoven.

6. Follow proper procedures for disposal of contaminated materials.





structo

r’s Gu

ide

3. what is the purpose of the control plates? Explain the difference between each and why it is necessary to run each.

Control plates help interpret the experimental results. There are three control plates in this experiment.

• Thecontrolplatethatislabeled“PLUSAMPControl”showsthat cells without the plasmid will not grow in the presence of ampicillin.

• Thecontrolplatelabeled“NOAMPControl”showsthatthecells without the plasmid are able to grow on agar without ampicillin.

• Theplate“NOAMPpBR322DNA”showsthatthecellswerenotdamaged during the transformation process and therefore are able to grow on agar plates that do not contain ampicillin.





stru

cto

r’s

Gu

ide

idealized Schematic of Results - protocol A and B

nO Amp plated with control cells

- pBR322 DnA

Result: white colo-nies. May look like a smeared layer of cells referred to as a "lawn".

Demonstrates: Original untransformed cells are viable in the absence of ampicillin.

plus Amp plated with control cells

- pBR322 DnA

Result: No growth

Demonstrates: Cells are sensitive to ampicillin. Without pBR322 DNA they are not ampicillin-resistant.

nO Amp platedwith cells

+ pBR322 DnA

Result: Both trans-formed and untrans-formed cells will grow as white colo-nies. May look like a smeared layer of cells.

Demonstrates: Trans-formed cells are viable in the absence of ampicillin.

plus Amp plated with cells

+ pBR322 DnA

Result: individual white colonies.

Demonstrates: Only transformed cells which are resistant to ampicillin due to the uptake of pBR322 DNA will grow.

NO AMPControl

White colonies

PLUS AMPControl

No colonies

NO AMPpBR322 DNA

White colonies

PLUS AMPpBR322 DNA

White colonies




structo

r’s Gu

ide

1. Exogenous DnA does not passively enter non-competent E. coli cells. what treatment do cells require to be competent?

E. coli can be artificially induced to enter competency when they are treated with the chloride salts of the metal cations calcium, mag-nesium 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 not contain ampicillin?

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

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

A successful transformation will show colonies on the plate contain-ing ampicillin with cells plus plasmid DNA (PLUS AMP pBR322 DNA).

4. what are some reasons why transformation may not be successful?

Unsuccessful transformations could result due to many reasons. They include not adding the plasmid to the + DNA tube, not add-ing bacteria to the + DNA tube, improper timing of the heat shock step, or not adding reagents to induce competency, such as CaCl2 or induction solvent.

Study questions and Answers

StabilitySection 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 UseSteps 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 NoneNo None

Yes Splash or dust proof

Eye wash

Wear protective clothing and equipment to prevent contact.

EDVOTEK®

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

Standard. 29 CFR 1910.1200 Standard must be consulted forspecific 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 IManufacturer'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.

14676 Rothgeb DriveRockville, MD 20850

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

Other Limits Recommended % (Optional)

(301) 251-5990

(301) 251-5990

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 CharacteristicsFlash Point (Method Used)

Extinguishing Media

Flammable Limits UELLEL

Melting Point

Evaporation Rate(Butyl Acetate = 1)

Specific Gravity (H 0 = 1) 2

Ampicillin

AmpicillinCAS# 7177-48-2 No data

No data

No data

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 spilledmaterial with water streams.

Avoid breathing vapors.

07/01/03

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.

14676 Rothgeb Drive Rockville, MD 20850

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

Other Limits Recommended % (Optional)

(301) 251-5990

(301) 251-5990

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

Solvent for Induction of Competency

05-25-05

None ------------------- Not established ------------------

No data No data No data

No data No data No data

Soluble Clear liquid

No data No data No data Use water spray, alcohol foam, dry chemical, or carbon dioxide

Wear protective equipment and SCBA with full facepiece. Move container from fire area if possible. Vapors may flow along surfaces and flash back.

EDVOTEK®

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 Avoid incompatibles Strong oxdizers

X

Inhalation/Ingestion: Nausea and vomiting Skin/eye contact: Rapid absorption causing irritation

Uniform or apron Avoid contact

Yes Yes Yes

None identified

Irritation Skin disorders

Wear protective clothing. Take up with sand or other absorbant and place in container Dispose of properly. Observe all federal, state, and local regulations.

Avoid contact None

SCBA

Yes None Yes None

Butyl rubber gloves Safety goggles

Sulfur dioxide, mercaptans, carbon monoxide, carbon dioxide, formaldehyde

Ingestion: Call medical help, do not induce vomiting Inhalation: remove to fresh air Skin/eye contact: Flush w/ water

Documents

R e i s e d a d n U p d a t e d - EDVOTEK · The Biotechnology Education Company® ® The Biotechnology Education Company ® • 1-800-EDVOTEK • U p d a t e d R e v i s e d a d