115

The Biotechnology Education Company ®

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

EVT 100202AM

EDVO-Kit

115Cancer GeneDetection

See Page 3 for storage instructions.

ExpErImEnT OBjECTIVE:

In this experiment, students will gain an understandingof the p53 tumor suppressor gene

and its role in familial cancers.

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

Cancer Gene Detection

EVT 100202AM

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.

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

EDVOTEK, The Biotechnology Education Company, and InstaStain are registered trademarks of EDVOTEK, Inc.. Ready-to-Load, UltraSpec-Agarose and FlashBlue are trademarks of EDVOTEK, Inc.

Page

Experiment Components 3

Experiment Requirements 3

Background Information 4

Experiment Procedures

Experiment Overview and General Instructions 9

Agarose Gel Electrophoresis 11

Study Questions 12

Instructor's Guidelines

Notes to the Instructor and Pre-Lab Preparations 13

Experiment Results and Analysis 19

Study Questions and Answers 20

Appendices 21

Material Safety Data Sheets 32

Table of Contents

�

115Experiment


EVT 100202AM

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

FAx: (�01) �40-058� • email: [email protected]

rEADy-TO-LOAD™ DnA sAmpLEs FOr ELECTrOphOrEsIs

A Standard DNA Fragments B Control DNA C Patient Peripheral blood DNA D Patient Breast Tumor DNA E Patient Normal Breast Tissue DNA

rEAGEnTs & suppLIEs

• UltraSpec-Agarose™ powder • Concentrated electrophoresis buffer • FlashBlue™ DNA Stain • InstaStain® Blue cards • Practice Gel Loading Solution • 1 ml pipet • Microtipped Transfer Pipets

Note: If you ordered Experiment #115-Q, the experiment components in-clude InstaStain® Ethidium bromide instead of FlashBlue™ and InstaStain® Blue DNA stains.

DNA samples are stable at room temperature. However, if the experiment will not be conducted within one month of receipt, it is recommended that the DNA samples be stored in the refrigerator.

DNA samples do not require heating prior to gel loading.

Experiment Components

requirements

• Horizontal gel electrophoresis apparatus • D.C. power supply • Automatic micropipets with tips • Balance • Microwave, hot plate or burner • Pipet pump • 250 ml flasks or beakers • Hot gloves • Safety goggles and disposable laboratory gloves • Small plastic trays or large weigh boats (for gel destaining) • DNA visualization system (white light) • Distilled or deionized water

4

Duplication of this document, in conjunction with use of accompanying reagents, is permitted for classroom/laboratory 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 © 1989,1992,1994,1997,1998, 2000, 2004, 2007, 2009, EDVOTEK, Inc., all rights reserved. EVT 100202AM

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

Cancer Gene Detection115 Experiment

Background Information

ABOuT FAmILy pEDIGrEEs

When drawing or studying a family pedi-gree, the following are general guidelines to the symbols used and their representa-tions:

• A Circle represents a female.• A square represents a male. • A shaded circle or square refers to a

person having some form of cancer. • An open (non-shaded) square or circle

represents a person who is free of cancer.

• A circle or square (either shaded or open) with a diagonal slash through it represents a person who is deceased.

In Li-Fraumeni syndrome, the pattern of cancers in family pedigrees suggest dominant inheritance. It is a genetic predisposition leading to specific types of cancers. Typically, the onset of cancer is at an early age, with multiple primary tumors.

Female free of cancer

Male free of cancer

Female with some form of cancer

Male with some form of cancer

Female deceased,

Male deceased,

or

or

CAnCEr GEnE DETECTIOn

Many contributory factors have been identified to cause the onset of cancers, that include exposure to certain carcinogens in our diets and environment. Several forms of cancer have familial predispositions. These cancers appear to be linked to inherited mutation of suppressor genes, such as p53.

Familial cancers constitute a very small fraction of the total reported cancers and they occur in dominant inher-ited patterns. Mutations that are directly inherited are referred to as germline mutations. Such mutations can be detected in familial pedigrees. A second type of mu-tation, known as somatic mutations, do not have direct genetic links and are acquired during the life of the indi-vidual. Patterns of typical hereditary and sporadically acquired nonhereditary pedigrees appear in Figure 1.

In a germline with an inherited mutation, a single somatic mutation within a suppressor gene will result in the inactivation of both alleles. By contrast, normal inherited suppressor genes, that are free of mutations, will require two sequential mutations to initiate tumors. This model is referred to as the "Two-hit" hypothesis.

HereditaryGermline Mutation

SporadicSomatic Mutation

Multiple Tumors Bilateral Tumors

Early Onset

SomaticMutation

Single Tumors Unilateral Tumors

Later onset

Normal Gene

First SomaticMutation

Second SomaticMutation

Figure 1: Hereditary and Sporadic models of gene inactivation.

5

Duplication of this document, in conjunction with use of accompanying reagents, is permitted for classroom/laboratory 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 © 1989,1992,1994,1997,1998, 2000, 2004, 2007, 2009, EDVOTEK, Inc., all rights reserved. EVT 100202AM


115Experiment



Historically, some of the first genes identified include the retinoblastoma (RB) gene, Wilm's’ tumor (WTI), neurofibromatosis type II gene and Li-Fraumeni syndrome. In Li-Fraumeni syndrome, a notable feature in family pedigrees, include a sarcoma patient and at least two immediate relatives with other cancers before the age of 45, as well as multiple cancers in other family members. This is illustrated in Figure 2.

With the advent of molecular biology applications to medicine, gene maps and the chromosomal locations of genes are becoming available as tools for the identification of predisposition for various diseases. The procedures used to obtain such information include DNA isolation and the analysis of point mutations in hot spot areas in cancer-related genes, such as p53. Several methods of analysis for the detection of point mutations in genes include DNA sequencing.

The Human genome project has provided information to link to the identification of many various cancers and other diseases to DNA sequence information. This infor-mation needs to be handled cautiously to assure confidentiality of patients’ genetic profiles.

The study of inherited cancers has given cancer molecular biologists the opportunity to search for genes that are critical in normal cell development and carcinogenesis. At the molecular level, cancer formation is characterized by alterations in both dominant oncogenes and tumor suppressor genes, such as p53. Suppressors are normal cellular proteins that are involved in limiting cell growth. By contrast, oncogenes are involved in promoting the growth of cells.

In recent years, the p53 tumor suppressor protein has become the center of many cancer biology studies. Be-cause it appears to be of major significance, there is great impetus to study how this gene functions in normal cells compared to cancer cells. The gene for the p53 protein is located on the short arm of chromosome 17. It encodes a 53,000 molecular nuclear phosphoprotein. Wild type (normal) p53 functions as a cell regulator. There is now well-documented evidence that normal p53 is a sequence-specific DNA-binding protein that is a transcriptional regu-lator. Upon introduction of mutations, p53 loses its ability to bind to DNA. By contrast, p53 that have mutations in specific hot spots promote uncontrolled cell growth and therefore function as oncogenes. For a tumor suppressor gene such as p53 to play a role in transformation in can-cer, both alleles need to be altered, as shown Figure 2.

Figure 2: Example of a Li-Fraumeni family pedigree.

BB.42

BR.36 SS.23BB.34

CN.2 CN.36

OS.13 LK.2

BB Bilateral breast cancerBR Breast cancerCN Brain tumorLK LeukemiaOS OsteosarcomaSS Soft tissue sarcoma

�






The p53 protein can be divided into three domains. The first is the amino terminus region which contains the transcriptional activation region. The second is the central region within the protein where the majority of critical "hot spot" mutations are located. These "hot spots" are sites where mutations are detected in high frequencies. They are between exons 5 through 8 where 95% of the mutations occur. Within this region there five subregions where point mutations are detected in human cancers. The third region of the p53 protein is the carboxyl section that is the most complex section that contains the oligomerization and nuclear localization sequences.

Examples of hot spots include codons 165 and 175 in exon 5; 196 and 213 in exon 6; 245 and 248 in exon 7; 273 and 282 in exon 8; all are within the p53 protein. Several of these mutations result in an altered p53 protein conformation. In turn, these changes can re-sult in increased stability of the mutant protein and the ability to bind to the normal p53 protein and inactivate it. It is of interest to note that there are correlations between the mutation and tumor tissue. One such example is the mutation at amino acid 175 which is common in colon carcinoma but is rarely observed in lung carcinoma. The inherited Li-Fraumeni syndrome as it has become to be known is rare. When it does occur it affects young family members and results in high mortality rates. Two physicians, Li and Fraumeni first described the syndrome after examining death certificates of 648 childhood sarcomas. It was discovered in four families where siblings and cousins had childhood sarcomas. Further analysis showed more than 50% of the affected families had extended phenotypes that included brain, breast cancers and leukemias. Cells in the individuals with LFS have a single wild type p53 allele. Examination of the p53 has shown a correlation to mutations in the protein as described above.

�



115Experiment


Constructing the Family pedigree

A first step in the search and assignment of Li-Fraumeni syndrome is to establish the family pedigree of the patient.

The first part of the experiment is based on the information made available as part of a diagnosis by the family physician and the oncologist. The pedigree information that you will develop is for a young woman who is suspected to have the Li-Fraumeni syndrome.

Upon monthly breast self-examination, Valerie Brown, age 36, found a small irregular mass. She was concerned because she knew that her mother had a mastectomy when she was in her late thirties. Valerie made an appointment with her physician, who referred her to a specialist at a local cancer center, where she was diagnosed as having breast cancer. As part of the medical work-up, the oncologist had inquired about her family history of cancer. Upon consultation with her mother, Valerie learned that her father and his family appeared to be free of cancer. However, in Valerie's mother's family, several cases of cancer have occurred.

With the information given below, chart the family pedigree.

• Her mother, Diane, was diagnosed and treated for breast cancer at the age of 39. • Valerie did not know that Diane had a sister, Mabel, who died at age 2 of a brain

tumor. • Diane's brother, James underwent surgery, followed by chemotherapy for colon

cancer. • Her maternal grandmother, Elsie, died at age 42 from bilateral breast cancer.• Her maternal grandfather, Elmer, was free of cancer and is 88 years old.• Her maternal cousin, Patrick (son of James), died of brain cancer at 14.• Her cousin, Jane, aged 2 who is Patrick's sister was diagnosed with childhood leu-

kemia and subsequently died.• Patrick's two other brothers, Robert, 28 and Curtis, 30, are in good health and free

of cancer. • Valerie's sister, Nancy is free of cancer.• Nancy's son, Michael was diagnosed at the age of 3 as having sarcoma. Recently,

at the age of 18, he was diagnosed as having osteosarcoma.• Nancy's other son, John, and daughter, Jessica, are free of cancer.

Valerie has five children: Justin (16), Sheila (14), Robert (10), Angela (8), and Anthony (6), none of whom show any signs of cancer at this time. She was interested in the p53 diagnostic test to determine if she inherited mutations.

8





Constructing the Family pedigree

The familial pedigree strongly suggests Li-Fraumeni syndrome. In such a case, a secondary diagnostic test is normally conducted. In this scenario, Valerie provides a sample of blood and tumor tissue to conduct DNA analysis for the p53 gene. Normally the procedure is to amplify the gene using polymerase chain reaction. This is followed by one of several methods to detect the presence of a point mutation at the hot spots.

In the simulation experiment which follows, Valerie's DNA has already been digested with a restriction enzyme that recognizes the mutant sequence at the simulated hot spot site at nucleotide 165 which is the palindrome CAGCTG. A restriction enzyme was used as a probe to cut the simulated amplified gene for Valerie’s DNA sample, together with a nor-mal control and a set of standard DNA marker fragments. Digestion of the normal ampli-fied DNA will give a characteristic DNA fragment banding pattern. The DNA obtained from blood lymphocyte will give an altered band pattern representing one normal allele and the second which is the mutant. The DNA analysis from the tumor tissue will show only the pattern for the tumor allele. The predigested samples with the control wild type and DNA markers will be separated by agarose gel electrophoresis and stained.

�



115Experiment

Cancer Gene DetectionExp

erimen

t proced

ure

Experiment Overview and General Instructions

ExpErImEnT OBjECTIVE:

In this experiment, students will gain an understanding of the p53 tumor suppressor gene and its role in familial cancers.

LABOrATOry sAFETy

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

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

3. DO NOT MOUTH PIPET REAGENTS - USE PIPET PUMPS.

4. Exercise caution when using any electrical equipment in the laboratory.

5. Always wash hands thoroughly with soap and water after handling reagents or biological materials in the laboratory.

LABOrATOry nOTEBOOK rECOrDInGs:

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

Before starting the Experiment:

• Write a hypothesis that reflects the experiment. • Predict experimental outcomes.

During the Experiment: • Record (draw) your observations, or photograph the results.

Following the Experiment: • Formulate an explanation from the results. • Determine what could be changed in the experiment if the experiment were repeated. • Write a hypothesis that would reflect this change.

10





Exp

erim

ent

pro

ced

ure

After electrophoresis, transfer gel for staining

Analysis on white

light source

FlashBlue™DNA stain

Attach safety cover,connect leads to power

source and conduct electrophoresis

Load eachsample in

consecutive wells

Remove end blocks & comb, then submerge

gel under buffer in electrophoresis

chamber

Prepare agarose gel in

casting tray

6

5

4

3

2

1

Gel pattern will vary depending upon experiment.

( - )

( + )

1 2 3 4 5 6

Experiment Overview: Flow Chart

11



115Experiment

Cancer Gene DetectionExp

erimen

t proced

ure

prepare the Gel

1. Prepare an agarose gel with specifications summarized below. Your instructor will specify which DNA stain you will be using.

• Agarose gel concentration required: 0.8%

• Recommended gel size: 7 x 7 cm or 7 x 14 cm (two gels)

• Number of sample wells required: 5

• Placement of well-former template: first set of notches ( 7 x 7 cm) first & third set of notches (7 x 14 cm)

Reminders:

During electrophoresis, the DNA samples migrate through the agarose gel to-wards the positive electrode. Before loading the samples, make sure the gel is properly oriented in the apparatus chamber.

+Black Red

Sample wells

–

Agarose Gel Electrophoresis

Lane Tube

1 A Standard DNA Fragments 2 B Control DNA 3 C Patient Peripheral blood DNA 4 D Patient Breast Tumor DNA 5 E Patient Normal Breast Tissue DNA

run the Gel

3. After DNA samples are loaded, connect the apparatus to the D.C. power source and set the power source at the required voltage.

4. Check that current is flowing properly - you should see bubbles form-ing on the two platinum electrodes. Conduct electrophoresis for the length of time specified by your instructor.

5. After electrophoresis is completed, proceed to DNA staining and visu-alization. Refer to Appendix E, F, G, or H for the appropriate staining instructions.

6. Document the results of the gel by photodocumentation.

Alternatively, place transparency film on the gel and trace it with a per-manent marking pen. Remember to include the outline of the gel and the sample wells in addition to the migration pattern of the DNA bands.

For gels to be stained with FlashBlue™ or InstaStain® Blue, prepare gels accord-ing to Appendix A.

For gels to be stained with InstaStain® Ethidium bromide, prepare gels ac-cording to Appendix B.

Step-by-step guidelines for agarose gel prepara-tion are summarized in Appendix D.

Wear Gloves & goggles

Load the samples

2. Load the DNA samples in tubes A - E into the wells in consecutive order.

• For gels to be stained with FlashBlue™ or InstaStain® Blue, fill wells with 35 - 38 µl.

• For gels to be stained with InstaStain® Ethidium Bromide, fill wells with 18 - 20 µl.

material safety Data sheetsFull-size (8.5 x 11”) pdf copy of MSDS is available at www. edvotek.com or by request.115

Experiment

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Oth

er L

imit

s R

eco

mm

end

ed%

(O

pti

on

al)

(301

) 25

1-59

90

(301

) 25

1-59

90

Bo

ilin

g P

oin

t

Sect

ion

III -

Ph

ysic

al/C

hem

ical

Ch

arac

teri

stic

s

Un

usu

al F

ire

and

Exp

losi

on

Haz

ard

s

Spec

ial F

ire

Fig

hti

ng

Pro

ced

ure

s

Vap

or

Pres

sure

(m

m H

g.)

Vap

or

Den

sity

(A

IR =

1)

Solu

bili

ty in

Wat

er

Ap

pea

ran

ce a

nd

Od

or

Sect

ion

IV -

Ph

ysic

al/C

hem

ical

Ch

arac

teri

stic

sFl

ash

Po

int

(Met

ho

d U

sed

)

Exti

ng

uis

hin

g M

edia

Flam

mab

le L

imit

sU

ELLE

L

Mel

tin

g P

oin

t

Evap

ora

tio

n R

ate

(Bu

tyl A

ceta

te =

1)

Spec

ific

Gra

vity

(H

0 =

1)

2

50x

Elec

tro

ph

ore

sis

Bu

ffer

This

pro

du

ct c

on

tain

s n

o h

azar

do

us

mat

eria

ls a

s d

efin

ed b

y th

e O

SHA

Haz

ard

Co

mm

un

icat

ion

Sta

nd

ard

.

No

dat

a

No

dat

a

No

dat

a

No

dat

a

No

dat

a

No

dat

a

Ap

pre

ciab

le, (

gre

ater

th

an 1

0%)

Cle

ar, l

iqu

id, s

ligh

t vi

neg

ar o

do

r

No

dat

a

N.D

. = N

o d

ata N.D

.

N.D

.

Use

ext

ing

uis

hin

g m

edia

ap

pro

pri

ate

for

surr

ou

nd

ing

fir

e.

Wea

r p

rote

ctiv

e eq

uip

men

t an

d S

CB

A w

ith

fu

ll fa

cep

iece

op

erat

ed in

po

siti

ve p

ress

ure

mo

de.

No

ne

iden

tifi

ed

10/0

5/06

Stab

ility

Sect

ion

V -

Rea

ctiv

ity

Dat

aU

nst

able

Sect

ion

VI -

Hea

lth

Haz

ard

Dat

a

Inco

mp

atib

ility

Co

nd

itio

ns

to A

void

Ro

ute

(s)

of

Entr

y:In

hal

atio

n?

Ing

esti

on

?Sk

in?

Oth

er

Stab

le

Haz

ard

ou

s Po

lym

eriz

atio

nM

ay O

ccu

rC

on

dit

ion

s to

Avo

id

Will

No

t O

ccu

r

Hea

lth

Haz

ard

s (A

cute

an

d C

hro

nic

)

Car

cin

og

enic

ity:

NTP

?O

SHA

Reg

ula

tio

n?

IAR

C M

on

og

rap

hs?

Sig

ns

and

Sym

pto

ms

of

Exp

osu

re

Med

ical

Co

nd

itio

ns

Gen

eral

ly A

gg

rava

ted

by

Exp

osu

re

Emer

gen

cy F

irst

Aid

Pro

ced

ure

s

Sect

ion

VII

- Pr

ecau

tio

ns

for

Safe

Han

dlin

g a

nd

Use

Step

s to

be

Take

n in

cas

e M

ater

ial i

s R

elea

sed

fo

r Sp

illed

Was

te D

isp

osa

l Met

ho

d

Prec

auti

on

s to

be

Take

n in

Han

dlin

g a

nd

Sto

rin

g

Oth

er P

reca

uti

on

s

Sect

ion

VIII

- C

on

tro

l Mea

sure

s

Ven

tila

tio

nLo

cal E

xhau

stSp

ecia

l

Mec

han

ical

(G

ener

al)

Res

pir

ato

ry P

rote

ctio

n (

Spec

ify

Typ

e)

Pro

tect

ive

Glo

ves

Oth

er P

rote

ctiv

e C

loth

ing

or

Equ

ipm

ent

Wo

rk/H

ygie

nic

Pra

ctic

es

Eye

Pro

tect

ion

Haz

ard

ou

s D

eco

mp

osi

tio

n o

r B

ypro

du

cts

X

N

on

e

Stro

ng

oxi

diz

ing

ag

ents

Car

bo

n m

on

oxi

de,

Car

bo

n d

ioxi

de

X

N

on

e

Yes

Y

es

Y

es

No

ne

No

ne

iden

tifi

ed

Irri

tati

on

to

up

per

res

pir

ato

ry t

ract

, ski

n, e

yes

No

ne

Ing

esti

on

: If

co

nsc

iou

s, g

ive

larg

e am

ou

nts

of

wat

er

Eyes

: Fl

ush

wit

h w

ater

In

hal

atio

n:

Mo

ve t

o f

resh

air

Sk

in:

Was

h w

ith

so

ap a

nd

wat

er

Wea

r su

itab

le p

rote

ctiv

e cl

oth

ing

. M

op

up

sp

ill

and

rin

se w

ith

wat

er, o

r co

llect

in a

bso

rpti

ve m

ater

ial a

nd

dis

po

se o

f th

e ab

sorp

tive

mat

eria

l.

Dis

po

se in

acc

ord

ance

wit

h a

ll ap

plic

able

fed

eral

, sta

te, a

nd

loca

l en

viro

men

tal r

egu

lati

on

s.

Avo

id e

ye a

nd

ski

n c

on

tact

.

No

ne

Yes

N

on

e

Yes

N

on

e

Yes

_Saf

ety

go

gg

les

No

ne

No

ne

Stab

ility

Sect

ion

V -

Rea

ctiv

ity

Dat

aU

nst

able

Sect

ion

VI -

Hea

lth

Haz

ard

Dat

a

Inco

mp

atib

ility

Co

nd

itio

ns

to A

void

Ro

ute

(s)

of

Entr

y:In

hal

atio

n?

Ing

esti

on

?Sk

in?

Oth

er

Stab

le

Haz

ard

ou

s Po

lym

eriz

atio

nM

ay O

ccu

rC

on

dit

ion

s to

Avo

id

Will

No

t O

ccu

r

Hea

lth

Haz

ard

s (A

cute

an

d C

hro

nic

)

Car

cin

og

enic

ity:

NTP

?O

SHA

Reg

ula

tio

n?

IAR

C M

on

og

rap

hs?

Sig

ns

and

Sym

pto

ms

of

Exp

osu

re

Med

ical

Co

nd

itio

ns

Gen

eral

ly A

gg

rava

ted

by

Exp

osu

re

Emer

gen

cy F

irst

Aid

Pro

ced

ure

s

Sect

ion

VII

- Pr

ecau

tio

ns

for

Safe

Han

dlin

g a

nd

Use

Step

s to

be

Take

n in

cas

e M

ater

ial i

s R

elea

sed

fo

r Sp

illed

Was

te D

isp

osa

l Met

ho

d

Prec

auti

on

s to

be

Take

n in

Han

dlin

g a

nd

Sto

rin

g

Oth

er P

reca

uti

on

s

Sect

ion

VIII

- C

on

tro

l Mea

sure

s

Ven

tila

tio

nLo

cal E

xhau

stSp

ecia

l

Mec

han

ical

(G

ener

al)

Res

pir

ato

ry P

rote

ctio

n (

Spec

ify

Typ

e)

Pro

tect

ive

Glo

ves

Oth

er P

rote

ctiv

e C

loth

ing

or

Equ

ipm

ent

Wo

rk/H

ygie

nic

Pra

ctic

es

Eye

Pro

tect

ion

Haz

ard

ou

s D

eco

mp

osi

tio

n o

r B

ypro

du

cts

X

N

on

e

No

ne

Sulf

ur

oxi

des

, an

d b

rom

ides

X

N

on

e

Yes

Y

es

Yes

Acu

te e

ye c

on

tact

: M

ay c

ause

irri

tati

on

. N

o d

ata

avai

lab

le f

or

oth

er r

ou

tes.

No

dat

a av

aila

ble

May

cau

se s

kin

or

eye

irri

tati

on

No

ne

rep

ort

ed

Trea

t sy

mp

tom

atic

ally

an

d s

up

po

rtiv

ely.

Rin

se c

on

tact

ed a

rea

wit

h c

op

iou

s am

ou

nts

of

wat

er.

Wea

r ey

e an

d s

kin

pro

tect

ion

an

d m

op

sp

ill a

rea.

Rin

se w

ith

wat

er.

Ob

serv

e al

l fed

eral

, sta

te, a

nd

loca

l reg

ula

tio

ns.

Avo

id e

ye a

nd

ski

n c

on

tact

.

No

ne

Yes

No

ne

Yes

No

ne

Yes

Spla

sh p

roo

f g

og

gle

s

No

ne

req

uir

ed

Avo

id e

ye a

nd

ski

n c

on

tact

Mat

eria

l Saf

ety

Dat

a Sh

eet

May

be

use

d t

o c

om

ply

wit

h O

SHA

's H

azar

d C

om

mu

nic

atio

nSt

and

ard

. 29

CFR

191

0.12

00 S

tan

dar

d m

ust

be

con

sult

ed f

or

spec

ific

req

uir

emen

ts.

IDEN

TITY

(A

s U

sed

on

Lab

el a

nd

Lis

t)N

ote

: B

lan

k sp

aces

are

no

t p

erm

itte

d.

If a

ny

item

is n

ot

app

licab

le, o

r n

o in

form

atio

n is

ava

ilab

le, t

he

spac

e m

ust

b

e m

arke

d t

o in

dic

ate

that

.

Sect

ion

IM

anu

fact

ure

r's

Nam

e

Sect

ion

II -

Haz

ard

ou

s In

gre

die

nts

/Id

enti

fy In

form

atio

n

Emer

gen

cy T

elep

ho

ne

Nu

mb

er

Tele

ph

on

e N

um

ber

fo

r in

form

atio

n

Dat

e Pr

epar

ed

Sig

nat

ure

of

Prep

arer

(o

pti

on

al)

Ad

dre

ss (

Nu

mb

er, S

tree

t, C

ity,

Sta

te,

Zip

Co

de)

EDV

OTE

K, I

nc.

1467

6 R

oth

geb

Dri

veR

ock

ville

, MD

208

50

Haz

ard

ou

s C

om

po

nen

ts [

Spec

ific

C

hem

ical

Iden

tity

; C

om

mo

n N

ame(

s)]

O

SHA

PEL

AC

GIH

TLV

Oth

er L

imit

s R

eco

mm

end

ed%

(O

pti

on

al)

(301

) 25

1-59

90

(301

) 25

1-59

90

Bo

ilin

g P

oin

t

Sect

ion

III -

Ph

ysic

al/C

hem

ical

Ch

arac

teri

stic

s

Un

usu

al F

ire

and

Exp

losi

on

Haz

ard

s

Spec

ial F

ire

Fig

hti

ng

Pro

ced

ure

s

Vap

or

Pres

sure

(m

m H

g.)

Vap

or

Den

sity

(A

IR =

1)

Solu

bili

ty in

Wat

er

Ap

pea

ran

ce a

nd

Od

or

Sect

ion

IV -

Ph

ysic

al/C

hem

ical

Ch

arac

teri

stic

sFl

ash

Po

int

(Met

ho

d U

sed

)

Exti

ng

uis

hin

g M

edia

Flam

mab

le L

imit

sU

ELLE

L

Mel

tin

g P

oin

t

Evap

ora

tio

n R

ate

(Bu

tyl A

ceta

te =

1)

Spec

ific

Gra

vity

(H

0 =

1)

2

Prac

tice

Gel

Lo

adin

g S

olu

tio

n

10/0

5/06

This

pro

du

ct c

on

tain

s n

o h

azar

do

us

mat

eria

ls a

s d

efin

ed b

y th

e O

SHA

Haz

ard

Co

mm

un

icat

ion

Stan

dar

d.

No

dat

a

No

dat

a

No

dat

a

No

dat

a

No

dat

a

No

dat

a

Solu

ble

B

lue

liqu

id, n

o o

do

r

No

dat

aN

o d

ata

No

dat

a

Dry

ch

emic

al, c

arb

on

dio

xid

e, w

ater

sp

ray

or

foam

Use

age

nts s

uita

ble

for t

ype

of su

rrou

ndin

g fir

e. K

eep

upw

ind,

avo

idb

reat

hin

g h

azar

do

us

sulf

ur

oxi

des

an

d b

rom

ides

. W

ear

SCB

A.

Un

kno

wn

ED

VO

TE

K®

��

115Experiment

material safety Data sheetsFull-size (8.5 x 11”) pdf copy of MSDS is available at www. edvotek.com or by request.

Stab

ility

Sect

ion

V -

Rea

ctiv

ity

Dat

aU

nst

able

Sect

ion

VI -

Hea

lth

Haz

ard

Dat

a

Inco

mp

atib

ility

Co

nd

itio

ns

to A

void

Ro

ute

(s)

of

Entr

y:In

hal

atio

n?

Ing

esti

on

?Sk

in?

Oth

er

Stab

le

Haz

ard

ou

s Po

lym

eriz

atio

nM

ay O

ccu

rC

on

dit

ion

s to

Avo

id

Will

No

t O

ccu

r

Hea

lth

Haz

ard

s (A

cute

an

d C

hro

nic

)

Car

cin

og

enic

ity:

NTP

?O

SHA

Reg

ula

tio

n?

IAR

C M

on

og

rap

hs?

Sig

ns

and

Sym

pto

ms

of

Exp

osu

re

Med

ical

Co

nd

itio

ns

Gen

eral

ly A

gg

rava

ted

by

Exp

osu

re

Emer

gen

cy F

irst

Aid

Pro

ced

ure

s

Sect

ion

VII

- Pr

ecau

tio

ns

for

Safe

Han

dlin

g a

nd

Use

Step

s to

be

Take

n in

cas

e M

ater

ial i

s R

elea

sed

fo

r Sp

illed

Was

te D

isp

osa

l Met

ho

d

Prec

auti

on

s to

be

Take

n in

Han

dlin

g a

nd

Sto

rin

g

Oth

er P

reca

uti

on

s

Sect

ion

VIII

- C

on

tro

l Mea

sure

s

Ven

tila

tio

nLo

cal E

xhau

stSp

ecia

l

Mec

han

ical

(G

ener

al)

Res

pir

ato

ry P

rote

ctio

n (

Spec

ify

Typ

e)

Pro

tect

ive

Glo

ves

Oth

er P

rote

ctiv

e C

loth

ing

or

Equ

ipm

ent

Wo

rk/H

ygie

nic

Pra

ctic

es

Eye

Pro

tect

ion

Haz

ard

ou

s D

eco

mp

osi

tio

n o

r B

ypro

du

cts

X

N

on

e

Stro

ng

oxi

diz

ing

ag

ents

Car

bo

n m

on

oxi

de,

Car

bo

n d

ioxi

de,

nit

rog

en o

xid

es, h

ydro

gen

bro

mid

e g

as

X

N

on

e

Yes

Y

es

Yes

No

dat

a av

aila

ble

Irri

tati

on

to

mu

cou

s m

emb

ran

es a

nd

up

per

res

pir

ato

ry t

ract

No

dat

a

Trea

t sy

mp

tom

atic

ally

an

d s

up

po

rtiv

ely

Wea

r SC

BA

, ru

bb

er b

oo

ts, r

ub

ber

glo

ves

Mix

mat

eria

l wit

h c

om

bu

stib

le s

olv

ent

and

bu

rn in

a c

hem

ical

inci

ner

ato

r eq

uip

ped

aft

erb

urn

er a

nd

scr

ub

ber

Use

in c

hem

ical

fu

me

ho

od

wit

h p

rop

er p

rote

ctiv

e la

b g

ear.

Mu

tag

en

Yes

Ch

em. f

um

e h

oo

d

No

N

on

e

Ru

bb

er

C

hem

. saf

ety

go

gg

les

R

ub

ber

bo

ots

Use

in c

hem

ical

fu

me

ho

od

wit

h p

rop

er p

rote

ctiv

e la

b g

ear.

Acu

te: M

ater

ial i

rrit

atin

g t

o m

uco

us

mem

bra

nes

, up

per

res

pir

ato

ry t

ract

, eye

s, s

kin

Ch

ron

ic:

May

alt

er g

enet

ic m

ater

ial

SCB

A

Mat

eria

l Saf

ety

Dat

a Sh

eet

May

be

use

d t

o c

om

ply

wit

h O

SHA

's H

azar

d C

om

mu

nic

atio

nSt

and

ard

. 29

CFR

191

0.12

00 S

tan

dar

d m

ust

be

con

sult

ed f

or

spec

ific

req

uir

emen

ts.

IDEN

TITY

(A

s U

sed

on

Lab

el a

nd

Lis

t)N

ote

: B

lan

k sp

aces

are

no

t p

erm

itte

d.

If a

ny

item

is n

ot

app

licab

le, o

r n

o in

form

atio

n is

ava

ilab

le, t

he

spac

e m

ust

b

e m

arke

d t

o in

dic

ate

that

.

Sect

ion

IM

anu

fact

ure

r's

Nam

e

Sect

ion

II -

Haz

ard

ou

s In

gre

die

nts

/Id

enti

fy In

form

atio

n

Emer

gen

cy T

elep

ho

ne

Nu

mb

er

Tele

ph

on

e N

um

ber

fo

r in

form

atio

n

Dat

e Pr

epar

ed

Sig

nat

ure

of

Prep

arer

(o

pti

on

al)

Inst

aSta

in, I

nc.

P.O

. Bo

x 12

32W

est

Bet

hes

da,

MD

208

27

Haz

ard

ou

s C

om

po

nen

ts [

Spec

ific

C

hem

ical

Iden

tity

; C

om

mo

n N

ame(

s)]

O

SHA

PEL

AC

GIH

TLV

Oth

er L

imit

s R

eco

mm

end

ed%

(O

pti

on

al)

(301

) 25

1-59

90

(301

) 25

1-59

90

Bo

ilin

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oin

t

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III -

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hem

ical

Ch

arac

teri

stic

s

Un

usu

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ire

and

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losi

on

Haz

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Spec

ial F

ire

Fig

hti

ng

Pro

ced

ure

s

Vap

or

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sure

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m H

g.)

Vap

or

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sity

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IR =

1)

Solu

bili

ty in

Wat

er

Ap

pea

ran

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nd

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or

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IV -

Ph

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al/C

hem

ical

Ch

arac

teri

stic

sFl

ash

Po

int

(Met

ho

d U

sed

)

Exti

ng

uis

hin

g M

edia

Flam

mab

le L

imit

sU

ELLE

L

Mel

tin

g P

oin

t

Evap

ora

tio

n R

ate

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tyl A

ceta

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1)

Spec

ific

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vity

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1)

2

Inst

aSta

in®

Eth

idiu

m B

rom

ide

Eth

idiu

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rom

ide

D

ata

no

t av

aila

ble

No

dat

a

No

dat

a

No

dat

a

No

dat

a

No

dat

a

No

dat

a

Solu

ble

Ch

emic

al b

ou

nd

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No

dat

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N.D

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o d

ata N.D

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.D.

Wat

er s

pra

y, c

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on

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10/0

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CA

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ED

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Mat

eria

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Dat

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wit

h O

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azar

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om

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atio

nSt

and

ard

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CFR

191

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00 S

tan

dar

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ust

be

con

sult

ed f

or

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ific

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uir

emen

ts.

IDEN

TITY

(A

s U

sed

on

Lab

el a

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Lis

t)N

ote

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lan

k sp

aces

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no

t p

erm

itte

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If a

ny

item

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ot

app

licab

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form

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n is

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ust

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ate

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.

Sect

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Nam

e

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II -

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tify

Info

rmat

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Emer

gen

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mb

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on

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um

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fo

r in

form

atio

n

Dat

e Pr

epar

ed

Sig

nat

ure

of

Prep

arer

(o

pti

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Ad

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Nu

mb

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Sta

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Zip

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EDV

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1467

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208

50

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om

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Spec

ific

C

hem

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Iden

tity

; C

om

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s)]

O

SHA

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AC

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(301

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(301

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hem

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Ch

arac

teri

stic

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Un

usu

al F

ire

and

Exp

losi

on

Haz

ard

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Spec

ial F

ire

Fig

hti

ng

Pro

ced

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s

Vap

or

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sure

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m H

g.)

Vap

or

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sity

(A

IR =

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Solu

bili

ty in

Wat

er

Ap

pea

ran

ce a

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or

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ion

IV -

Ph

ysic

al/C

hem

ical

Ch

arac

teri

stic

sFl

ash

Po

int

(Met

ho

d U

sed

)

Exti

ng

uis

hin

g M

edia

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mab

le L

imit

sU

ELLE

L

Mel

tin

g P

oin

t

Evap

ora

tio

n R

ate

(Bu

tyl A

ceta

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1)

Spec

ific

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0 =

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ED

VO

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IUM

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No

dat

a av

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CA

S #

61-7

3-4

No

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a

No

dat

a

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dat

a

No

dat

a

No

dat

a

No

dat

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Solu

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- c

old

Ch

emic

al b

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dat

a av

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No

dat

a

N

o d

ata

Wat

er s

pra

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on

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co

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bre

ath

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atu

s an

d p

rote

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to

pre

ven

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nta

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wit

h s

kin

an

d e

yes

Emit

s to

xid

fu

mes

un

der

fir

e co

nd

itio

ns

Stab

ility

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V -

Rea

ctiv

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Dat

aU

nst

able

Sect

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VI -

Hea

lth

Haz

ard

Dat

a

Inco

mp

atib

ility

Co

nd

itio

ns

to A

void

Ro

ute

(s)

of

Entr

y:In

hal

atio

n?

Ing

esti

on

?Sk

in?

Oth

er

Stab

le

Haz

ard

ou

s Po

lym

eriz

atio

nM

ay O

ccu

rC

on

dit

ion

s to

Avo

id

Will

No

t O

ccu

r

Hea

lth

Haz

ard

s (A

cute

an

d C

hro

nic

)

Car

cin

og

enic

ity:

NTP

?O

SHA

Reg

ula

tio

n?

IAR

C M

on

og

rap

hs?

Sig

ns

and

Sym

pto

ms

of

Exp

osu

re

Med

ical

Co

nd

itio

ns

Gen

eral

ly A

gg

rava

ted

by

Exp

osu

re

Emer

gen

cy F

irst

Aid

Pro

ced

ure

s

Sect

ion

VII

- Pr

ecau

tio

ns

for

Safe

Han

dlin

g a

nd

Use

Step

s to

be

Take

n in

cas

e M

ater

ial i

s R

elea

sed

fo

r Sp

illed

Was

te D

isp

osa

l Met

ho

d

Prec

auti

on

s to

be

Take

n in

Han

dlin

g a

nd

Sto

rin

g

Oth

er P

reca

uti

on

s

Sect

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VIII

- C

on

tro

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sure

s

Ven

tila

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cal E

xhau

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ecia

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Mec

han

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(G

ener

al)

Res

pir

ato

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rote

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Spec

ify

Typ

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tect

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Glo

ves

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rote

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loth

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ygie

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es

Eye

Pro

tect

ion

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ard

ou

s D

eco

mp

osi

tio

n o

r B

ypro

du

cts

X

No

ne

Stro

ng

oxi

diz

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ag

ents

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c fu

mes

of

Car

bo

n m

on

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de,

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bo

n d

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de,

n

itro

gen

oxi

des

, su

lfu

r o

xid

es, h

ydro

gen

, ch

lori

de

gas

X

N

on

e

Yes

Y

es

Yes

Skin

: M

ay c

ause

ski

n ir

rita

tio

n

Eyes

: M

ay c

ause

eye

irri

tati

on

In

hal

atio

n:

Cya

no

sis

Mee

ts c

rite

ria

for

pro

po

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OSH

A m

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rule

PER

EAC

47.

3042

0.82

No

dat

a av

aila

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dat

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Trea

t sy

mp

tom

atic

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Ven

tila

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rea

and

was

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pill

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Mix

mat

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l wit

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t an

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ore

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No

ne

MIO

SH/O

SHA

ap

pro

ved

, SC

BA

Req

uir

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hem

. saf

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go

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les

Ru

bb

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oo

ts

�4The Biotechnology Education Company® • 1-800-EDVOTEK • www.edvotek.com


EVT 100202AM

EDVOTEK series 100 Electrophoresis Experiments:

Cat. # Title

101 Principles and Practice of Agarose Gel Electrophoresis

102 Restriction Enzyme Cleavage Patterns of DNA

103 PCR - Polymerase Chain Reaction

104 Size Determination of DNA Restriction Fragments

105 Mapping of Restriction Sites on Plasmid DNA

109 DNA Fingerprinting - Identification of DNA by Restriction Fragmentation Patterns

112 Analysis of Eco RI Cleavage Patterns of Lambda DNA

114 DNA Paternity Testing Simulation

115 Cancer Gene Detection

116 Sickle Cell Gene Detection (DNA-based)

117 Detection of Mad Cow Disease

118 Cholesterol Diagnostiics

124 DNA-based Screening for Smallpox

130 DNA Fingerprinting - Amplification of DNA for Fingerprinting

Visit our web site for information about the above experiments and other products

in EDVOTEK’s comprehensive offerings for biotechnology and biology education.

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