Transformation With pBR32

The Biotechnology Education Company®
®
sed
Revi
and
ated
Upd
EDVO-Kit #
201
Transformation
of E. coli with pBR322
Storage: See Page 3 for

specific storage instructions
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.
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.
The Biotechnology Education Company ® • 1-800-EDVOTEK • www.edvotek.com
EVT 091102K
2 EDVO-Kit # 201 Transformation of E. coli with pBR322
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

EVT 091102K
Transformation of E. coli with pBR322 EDVO-Kit # 201 3
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.
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
Protocol A Protocol B
There are subtle differ-
Conventional AP Biology
ences between the two
protocols provided with Method Lab 6A
this experiment. The
major difference be-
Instructor’ Pre-lab Prep

Incubate vial
tween the two options of cells
overnight at 34°C
are illustrated in the Resuspended
figure at right. Freeze-dried
Instructor’ Pre-lab Prep
Plate cells and grow

E. coli cells overnight at 37°C
Add Competency
Induction Solvent
and Incubate on ice
for 30 minutes
Students begin
Aliquot for Experiment
Protocol B
cells
each group
0.7 ml
Cell Suspension
+DNA
Students
transfer 2-3
-DNA
large colonies
250 µl
CaCl2
Students begin
Experiment
Protocol A
Control
Buffer
pBR322
DNA
Students
transfer
300 µl Cells
After choosing a protocol option, perform all pre-lab and experimental

procedures consistently in accordance with instructions for the chosen
protocol.
EDVOTEK - The Biotechnology Education Company ®

1-800-EDVOTEK • www.edvotek.com
24-hour FAX: (301) 340-0582 • email: edvotek@aol.com
EVT 091102K
Experiment Components

Storage: Store components A-G in the refrigerator.
Component
A Transformation LyphoCells™ (DO NOT FREEZE)
Quantities: B Supercoiled pBR322 DNA
C Control Buffer (no DNA) (not used in Protocol B)
Experiment # 201 is D Ampicillin
designed for 10 groups E Cell reconstitution medium
(2-4 students per F Solvent for induction of competency (not used in Protocol B)
group). G CaCl2 (not used in Protocol A)
Storage: Store components listed below at Room temperature

• Bottle ReadyPour™ Luria Broth Agar, sterile
(also referred to as ReadyPour medium)
• Bottle Luria Broth Medium for Recovery, 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
All components are

intended for educational
research only. They are
not to be used for diag-
nostic or drug purposes, Important READ ME!
nor administered to or
consumed by humans or In this experiment, antibiotics are used for the selection of transformed bac-
animals. teria. Students who have or suspect to have allergies to penicilllin, ampicil-
lin or tetracycine should not participate in this experiment.
None of the experiment
components are derived
from human sources.

EVT 091102K
Requirements
• Automatic Micropipet (5-50 µl) and tips

• Two Water baths* (37°C and 42°C)
• Thermometer
• Incubation Oven
• Pipet pumps or bulbs
• Marking pens
• Bunsen burner, hot plate or microwave oven
• Hot gloves
• 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.
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EVT 091102K
Bacterial Transformation
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
Background Information
DNA was the genetic material.
The transformation process involves the

uptake of exogenous DNA by the cell
Number of final vol at which results in a newly acquired genetic
Number of trait that is stable and heritable. Bacteri-
transformants X recovery (ml) =
transformants al cells must be in a particular physiologi-
µg of DNA vol plated (ml)
per µg cal state before they can be transformed.
This state is referred to as competency.
Specific example: Competency can occur naturally in cer-
tain species of Haemophilus and Bacillus
100 100,000
1 ml when the levels of nutrients and oxy-
transformants X = (1 x 105)
gen are low. Competent Haemophilus
0.01 µg 0.1 ml transformants
express a membrane associated trans-
per µg
port complex which binds and transfers
certain DNA molecules from the medium
Figure 1: into the cell where they are incorporated
Bacterial Transformation Efficiency Calculation 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
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
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
Background Information
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
Relaxed limited growth in the zones that have been cleared of ampicil-
Supercoiled
lin. Colonies consisting of untransformed cells typically form small
colonies that are called satellites since they only appear around the
Figure 2: Supercoiled and larger transformed colonies. Larger plating volumes of cells and lon-
circular forms of plasmid DNAs 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.
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.
The Experiment
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.
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.
The 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:
• Autoclave at 121° C for 20 minutes.

Tape several petri plates together and close tube caps before
disposal. Collect all contaminated materials in an autoclavable,
disposable bag. Seal the bag and place it in a metal tray to pre-
vent any possibility of liquid medium or agar from spilling into
the sterilizer chamber.
• Soak in 10% bleach solution.

Immerse petri plates, open tubes and other contaminated 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.
Protocol A - Conventional Method Using EDVOTEK Lyphocells™
Transformation Experiment Flow Chart
0.7 ml
Cells
0.25 ml 0.25 ml
The Experiment
Cells Cells
25 µl Control 25 µl
Buffer pBR322 DNA
Control pBR322
Incubate on ice for
Buffer DNA
10 minutes
Incubate at 42°C
for 90 seconds
Place on ice
for 1 minute
Add 0.75 ml recovery broth
Incubate in 37°C
water bath for 30 minutes
For optimal results, store

covered plates in the up- Streak 0.25 ml of cell
suspension on plates
right position after streaking
to allow the cell suspen-
sion to be absorbed by the
agar. After approximately
20 minutes, invert the plates NO AMP PLUS AMP NO AMP PLUS AMP
for overnight incubation at Control Control pBR322 DNA pBR322 DNA
37°C. Incubate Incubate
overnight in 37°C overnight in 37°C
incubation oven incubation oven
NO AMP PLUS AMP NO AMP PLUS AMP

Control Control pBR322 DNA pBR322 DNA
White Colonies No Colonies White Colonies White Colonies

Plates contaning antibiotic have a stripe on the side of the plate.
Setting up the Transformation and Control

Experiment
1. Put your initials or group number on the
tubes labeled “pBR322 DNA” (contains 25 µl
pBR322
DNA
of plasmid DNA) and “Control Buffer” (con-
Control
Buffer
The Experiment
tains 25 µl of buffer) . Place them back on ice.
2. Set up the Control:

• Using a sterile transfer pipet,
transfer 0.25 ml (250 µl) of the cell
suspension to the tube “Control
Buffer”.
• Carefully place the pipet back into
the wrapper.
22
Bufftrol
er
DN3A
• Cap the tube; mix by tapping. Put
pBR
Con
the tube back on ice.
3. Set up the transformation:

• Using the same pipet from Step 2, transfer 0.25 ml (250 µl)
of the cell suspension to the tube “pBR322 DNA”.
• Cap the tube; mix by tapping. Put the tube back on ice.
1 ml
0.75 ml 4. Incubate the cells prepared in steps 1 - 3 on ice

for 10 minutes.
+ DNA
- DNA
0.5 ml
5. Place both tubes in a waterbath at

0.25 ml 42°C for 90 seconds. 42°C
+ DNA
This heat shock step facilitates the

- DNA
~ 0.06 ml entry of DNA in bacterial cells.
< 0.02 ml
6. Return both tubes to incubate on ice

Guide for Sterile for for 1 minute.
Calibrated
Transfer Pipet
+ DNA
- DNA
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.
The Experiment
8. Add 0.75 ml of the Recovery Broth
Control
Buffer
to the tube “pBR322 DNA”.
pBR322
DNA
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. 37°C
+ DNA
- DNA
9. Incubate the closed tubes in a 37°C water
bath for 30 minutes for a recovery pe-
riod.
10. While the tubes are incubating, label 4 agar plates as indicated
below. Write on the bottom or side of the petri plate.
• Label one unstriped plate: NO AMP Control

• Label one unstriped plate: NO AMP pBR322 DNA

• Label one striped plate: PLUS AMP Control
• Label one striped plate: PLUS AMP pBR322 DNA
• Put your initials or group number on all the plates.

pBR322
DNA
Control
Buffer
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.
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:
The Experiment

• 0.25 ml to the plate labeled NO AMP Control
• 0.25 ml to the plate labeled PLUS AMP Control
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 Same plate - To avoid contamination when plating, do not

one direction spread cells 90° set the lid down on the lab bench -- Lift the
to first direction lid of the plate only enough to allow spread-
ing. Be careful to avoid gouging the loop
into the agar.
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:

• 0.25 ml to the plate labeled NO AMP pBR322 DNA
• 0.25 ml to the plate labeled PLUS AMP pBR322 DNA

16. Spread the cells with a sterile inoculating loop.

Reminder:
Follow proper
17. Cover the plate and allow the liquid to be absorbed (approximately
procedures
15-20 minutes).
for disposal of
contaminated
materials.
Preparing Plates for Incubation

18. Stack your group's set of plates on top of one another and tape
them together.
The Experiment
19. Put your initials or group number on the taped set of plates.
If you do not have an incu-

bation oven, the plates can 20. Place the set of plates in a safe place designated by your instructor.
be left at room tempera- The plates should be left in the upright position to allow the cell
suspension to be absorbed by the agar for 15 - 20 minutes.
ture. Colonies of trans-
formed cells should appear
between 24 - 48 hours. 21. Place the plates in the inverted position (agar
side on top) in a 37°C bacterial incubation
oven for overnight incubation (15-20 hours).
If the cells have not been absorbed into the
Group 4
Reminder: medium, it is best to incubate the plates upright.
Follow proper The plates are inverted to prevent condensa-
procedures tion on the lid, which could drip onto the
for disposal of culture and interfere with experimental results.
contaminated
materials.
Viewing Plates After Incubation
22. Proceed to analyzing your results.
23. After analyzing your results, follow proper procedures for disposal
of contaminated materials.
Protocol B - Advanced Placement Biology Lab 6A
Transformation Experiment Flow Chart
Prepare Source Plates

DAY BEFORE LAB
E.coli cells
The Experiment
* The 37°C incubation for
30 minutes (after adding Transfer 2-3
large colonies
the Recovery Broth) is not
described in the AP Biology
Student Laboratory Manual
- DNA
+ DNA
+ 10 µl
(1997 Edition D). However, pBR322
DNA
EDVOTEK recommends 0.25 ml (pAMP)
that you perform this step CaCl 2
to allow the cells to recov-

Incubate on ice
er and begin to express the for 15 minutes
antibiotic resistance genes.
The Recovery Broth does Incubate at 42°C
for 90 seconds
not contain antibiotic.
Incubate on ice
for 2 minutes
Add 0.25 ml
Luria
+ DNA
- DNA
Recovery Broth
37°C for
Streak 0.25 ml of 30 minutes Streak 0.25 ml of
- DNA cells + DNA cells
on each plate on each plate

Plates contaning
Incubate antibiotic have a stripe
Incubate
overnight in 37°C on the side of the plate. overnight in 37°C
incubation oven incubation oven

Setting up the Transformation and Control

Experiment
Reminder 1. Label one microcentrifuge tube “ + DNA “. This will be the trans-
formation tube with plasmid DNA.
Contains
“+” plasmid DNA
The Experiment
2. Label a second microcentrifuge tube “
- DNA
Does not contain - DNA “. This will be the experimental
+ DNA
“-” plasmid DNA control tube without plasmid DNA.
3. Using a sterile 1 ml pipet, add 250 µl

(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 “:
• use a sterile toothpick to transfer 2 colonies (2-4 mm) from the
source plate to the test tubes.
Source Plate
E. coli Cells • Between your fingers, twist the toothpick vigorously and up
and down in the CaCl2 solution to dislodge and emulsify the
cells.
Transfer 2-4 large

colonies to each 5. Suspend the cells in both tubes by
tube containing pBR322 tapping or vortexing.
ice cold
CaCl2
+
+ DNA
- DNA
10 µl
250 µl 6. Add 10 µl of pBR322 DNA to the
CaCl2 tube labeled “ + DNA “.
This plasmid contains the ampicillin

Avoid scraping up agar when transferring the cells resistance gene, often referred to
from the source plate to the tubes with calcium as pAMP.
chloride solution. It is important that the cells are
resuspended in the calcium chloride solution and is 7. Incubate the two
not left on the toothpick or on the wall of the tube. tubes on ice for
+ DNA
- DNA
15 minutes.
8. Place both tubes in a waterbath at 42°C

for 90 seconds for the heat shock step.
42°C
This facilitates the entry of DNA in bacte-
+ DNA
- DNA
rial cells.
The Experiment
9. Return both tubes im-
mediately to the ice
+ DNA
- DNA
bucket and incubate for

two minutes.
10. Using a sterile pipet, add 250 µl (0.25 ml) of Recovery Broth to
each tube and mix.
- DNA
+ DNA
11. Incubate the cells for 30 minutes in a 37°C waterbath for a

recovery period.*
37°C
* This step (#11) is not described in the
AP Biology Student Laboratory Manual
+ DNA
- DNA
(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.
12. While the tubes are incubating, label 4 agar plates as indicated
below. Write on the bottom or side of the petri plate.
• Label one unstriped plate: NO AMP Control

• Label one unstriped plate: NO AMP pBR322 DNA

• Label one striped plate: PLUS AMP Control
• Label one striped plate: PLUS AMP pBR322 DNA
• Put your initials or group number on all the plates.
13. After the recovery period, remove the tubes

+ DNA
- DNA
from the water bath and place them on

the lab bench. Proceed to plating the
cells for incubation.
Plating the cells
Plating cells from the tube labeled " - DNA "
Reminder 14. Use a sterile 1 ml pipet to transfer recovered cells from the tube
labeled " - DNA " to the middle of the following plates:
The Experiment
Contains
“+” plasmid DNA • 0.25 ml to the plate labeled NO AMP Control
• 0.25 ml to the plate labeled PLUS AMP Control
Does not contain
“-” plasmid DNA
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 Same plate:
spreading. Be careful Spread cells spread cells 90° to
to avoid gouging the in one direction first direction
loop into the agar.
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:

• 0.25 ml to the plate labeled NO AMP pBR322 DNA
• 0.25 ml to the plate labeled PLUS AMP pBR322 DNA

18. Spread the cells with a sterile inoculating loop.

19. Cover the plate and allow the liquid to be absorbed (approximately
15-20 minutes).
.
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.
The Experiment
If you do not have an incu-
bation oven, the plates can 20. Place the set of plates in a safe place designated by your instructor.
be left at room tempera- The plates should be left in the upright position to allow the cell
suspension to be absorbed by the agar for 15 - 20 minutes.
ture. Colonies of trans-
formed cells should appear
between 24 - 48 hours. 21. Place the plates in the inverted position (agar
side on top) in a 37°C bacterial incubation
oven for overnight incubation (15-20 hours).
Group 4
If the cells have not been absorbed into the
Reminder: medium, it is best to incubate the plates upright.
Follow proper The plates are inverted to prevent condensation
procedures on the lid, which could drip onto the culture and
for disposal of may interfere with experimental results.
contaminated
materials.
Viewing Plates After Incubation
22. Proceed to analyzing your results.
23. After analyzing your results, follow proper procedures for disposal
of contaminated materials.
Laboratory notebook recordings:
Address and record the following in your laboratory notebook or on a

separate worksheet.
Before starting the Experiment:

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.
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
Data Collection
1. Observe the results you obtained on your transformation and con-

trol plates.
Transformation Plate: + DNA
The Experiment
• NO AMP pBR322 DNA = LB medium with cells transformed
with plasmid
• PLUS AMP pBR322 DNA = LB medium containing ampicillin

with cells transformed with plasmid
Control Plates: - DNA
• NO AMP Control = LB medium with cells NOT transformed with

plasmid
• PLUS AMP Control = LB medium containing ampicillin with cells

NOT transformed with plasmid
2. Draw and describe what you observe. For each of the plates, record
the following:

• How much bacterial growth do you observe? Determine a
count.
• What color are the bacteria?
• Why do different members of your class have different transfor-
mation efficiency values?
• If you did not get any results, what factors could be attributed
to this fact?
Determination of Transformation Efficiency

Transformation efficiency is a quantitative determination of how many
cells were transformed per 1 µg of plasmid DNA. In essence, it is an indi-
cator of how well the transformation experiment worked.
The Experiment
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:
Number of final vol at

Number of
transformants recovery (ml)
x = transformants
µg of DNA vol plated (ml) per µg
Example: Assume you observed 40 colonies: Quick Reference for

Expt. 201 - Protocol A:
40 6400
transformants 1.0 ml (6.4 x 103) 25 ng (0.025 µg) of DNA is used.
x =
0.025 µg 0.25 ml transformants
per µg 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.
Study Questions
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?
The Experiment
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?

EVT 091102K
Instructor’s Guide
Notes to the Instructor:
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
Online Ordering
are factors which must be considered in the planning and the implemen-
now available 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
Visit our web site for 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,
information about
Eastern time zone. Call 1-800-EDVOTEK for help from our knowledge-
EDVOTEK's complete able technical staff.
line of experiments for
biotechnology and Day 1: (Prior to the Lab)
biology education.
Technical Service • Prepare agar plates
Department • Prepare E. coli Cells (overnight incuba-
tion).
C H S E RV I C E • Dispense the DNA and control buffer
O -TE Mon - Fri
E DV
9:00 am to 6:00 pm ET Day 2: (Day of Lab Experiment)
1-800-EDVOTEK FAX: (301) 340-0582
(1-800-338-6835)
• Equilibrate water baths at 37°C and
web: www.edvotek.com 42°C; incubation oven at 37°C
email: edvotek@aol.com • Students transform cells and plate for
ET
overnight incubation.
m
Mo - 6p
n - Fri 9 am Please have the following information: Day 3: (Day after Lab Experiment)
• The experiment number and title
• Kit Lot number on box or tube • Students observe transformants and
• The literature version number controls
(in lower right corner) • Students calculate transformation ef-
• Approximate purchase date ficiency
• Follow clean up and disposal procedures
as outlined in the Laboratory Safety sec-
tion.

EVT 091102K
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:
• Reconstitute and incubate the LyphoCells™ at 37°C for 16 to 24
hours before the laboratory (overnight incubation).
• Competent cells must be dispensed just prior to the lab experiment.
If tubes are already labeled, dispensing will require approximately
15 minutes.
If performing Protocol B:
• Reconstitute the LyphoCells™, plate for individual colonies, and
incubate at 37°C for 16 to 24 hours before the laboratory (overnight
incubation).
LABORATORY
NOTEBOOKS For BOTH Protocols A and B:
It is highly recommended 1. The agar plates can be prepared several days in advance and
that students maintain a stored inverted (agar side on top) in the refrigerator. Prepara-
laboratory notebook to tion requires approximately 1 hour.
formulate hypotheses and
to record experimental 2. Dispensing the DNA and reagents requires approximately 30
procedures and results. minutes. This can be done the day before the lab and stored in
the refrigerator.
• EDVOTEK Cat. # 1401,
Laboratory DataBook 3. Allow ample time for the equilibration of the water baths at
is recommended. 37°C and 42°C and a bacterial incubation oven at 37°C on the
• Guidelines for keeping day of the experiment.
a laboratory notebook
is available at the 4. In the experiment, each group will perform the transformation
EDVOTEK web site. 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. Colonies will also appear between 24 - 48 hours at room
temperature.
Pour Agar Plates

(Prior to the Lab experiment)
• For optimal results, prepare plates two days prior to plating and set
aside the plates inverted at room temperature.
• If they are poured more than two days before use, they should be
stored inverted in the refrigerator. Remove the plates from the
Wear Hot Gloves
refrigerator and store inverted for two days at room temperature
and Goggles during all before use.
steps involving heating.
Heat the ReadyPour™ Medium
1. Equilibrate a water bath at 60°C for step 5 below.
2. Loosen, but do not remove, the cap on 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:
• Heat the bottle on High for two 30 second intervals.
• Using a hot glove, swirl and heat on High for an additional
25 seconds, or until all the medium is dissolved.
• Using a hot glove, occasionally swirl to expedite melting.
B. Hot plate or burner method:

• Place the bottle in a beaker partially filled with water.
• Heat the beaker to boiling over a hot plate or burner.
• Using a hot glove, occasionally swirl to expedite melting.
5. Allow the melted ReadyPour medium to cool. Placing the bottle in

a 60°C water bath will allow the agar to cool, while
preventing it from prematurely solidifying.
When the ReadyPour™ medium reaches approximately 60˚C

60°C, the bottle will be warm to the touch but not burn-
ing hot.
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.
• Open one sleeve of 20 plates and stack the plates neatly.

• Start the marker at the bottom of the stack and move the
marker vertically to the top plate to "stripe" the sides of the 20
plates.
• These plates will be used for medium with ampicillin.
• Do not stripe the second sleeve of plates. These will be the
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.
Quick Reference: Pouring Agar Plates
• 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.
9. Add the entire amount of ampicillin powder to the remaining mol-

ten ReadyPour medium in the bottle.
Add reagents to me-
dium which has been 10. Recap the bottle and swirl to completely dissolve the ampicillin.
cooled. Hot medium 11. Pour 20 transformation plates
will cause reagents
(with ampicillin, striped plates):
such as ampicillin to
rapidly decompose. 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.
Reminder:
Follow proper If you have extra sterile petri plates on hand, use any remaining me-
procedures dium to pour additional plates for the optional activity described on
for disposal of page 34.
contaminated
materials.
Summary of Poured Plates

Summary of Poured Plates Protocol B
Protocol A
5 source plates - large plates:

20 control plates - small no stripe 10 ml each - ReadyPour medium
plates: 5 ml each - ReadyPour
medium (no ampicillin) 20 control plates - small no stripe
20 transformation plates - small striped medium (no ampicillin)
medium with ampicillin 20 transformation plates - small striped
medium with ampicillin
Pre-Lab Preparations - Protocol A
Day before the experiment

Add 6 ml
Preparation of E. coli Cells
reconstitution
media (E) to
1. Use a 10 ml sterile pipet to add 6 ml cell reconstitu-
LyphoCells
tion medium (E) to the vial of LyphoCells.
Incubate vial of cells 2. Replace the rubber stopper and cap. Mix by gently
overnight at 37°C inverting until the freeze dried plug is dissolved.
3. Incubate the vial at 37°C for 16 - 24 hours (overnight)

in an incubation oven. For optimal results, incubate
LyphoCells at 37°C for 19 hours.
Place vial of
cells on ice
10 minutes Day of the experiment
Preparation of E. coli Cells, continued
Add 3 ml 4. Completely thaw the competency

ice cold induction solvent (F) and place on The solvent
Competency ice. (If there is a white precipitate for induction
Induction in the bottle, warm it in a 37°C wa- of competency
Solvent (F) terbath to dissolve the precipitate.) contains salts
5. Mix and resuspend the vial of incu- to make cells
bated cells by inverting and gently competent.
shaking. Place the vial on ice for 10
Incubate on ice minutes.
30 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
Dispense the walls of the pipet.
Cells
0.7 ml
Cell 7. Mix the cells and induction solvent thoroughly by
Suspension inverting the vial several times. The solution should
have no dense layers, "streams" or globules (i.e. it
should be a uniform suspension).
Students begin 8. Keep the cells on ice for a minimum of 30 minutes.

Experiment Cells can be kept on ice for up to 3 hours.
Protocol A
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-

pense 25 µl of the supercoiled pBR322
Summary of Reagent Preparations DNA to each of 10 microtest tubes
labeled "pBR322 DNA".
20 plates NO AMP (No Stripe) 5 ml
20 plates PLUS AMP (Stripe) 5 ml 17. Cap the tubes and place them on ice.
10 tubes pBR322 DNA 25 µl on ice
10 tubes Control Buffer 25 µl on ice 18. Using a FRESH micropipet tip, dispense
10 tubes Cells 0.7 ml on ice 25 µl of control buffer to each of 10 mi-
crotest tubes labeled "Control Buffer".
10 tubes Recovery Broth 1.5 ml
19. Cap the tubes and place them on ice.
Each Group Requires: Recovery Broth
1 tube pBR322 DNA 25 µl on ice Set up a classroom pipeting station, or dis-

1 tube Cells 0.7 ml on ice pense recovery broth (optional).
1 tube Control Buffer 25 µl on ice
2 striped plates 20. Dispense 1.5 ml Recovery Broth into 10
2 unstriped plates sterile tubes labeled "Recovery Broth"
4 sterile 1 ml pipets using a sterile pipet.
2 sterile inoculating loops 21. Cap the tubes and place them in the re-
1 tube Recovery broth (optional) 1.5 ml frigerator if not to be used immediately.
Pre-Lab Preparations - Protocol B
Day before the experiment
Add 6 ml This experiment requires preparation of isolated E.coli host

reconstitution transformation colonies 16-24 hours before the laboratory ex-
media (E) to periment, so plan accordingly.
LyphoCells vial
Important: Do not prepare source plates more than 24 hours
Incubate cells before the experiment. Old source plates will compromise the
30 - 60 minutes at 37°C success of the transformation experiment.
Preparation of E. coli Cells

Transfer 0.3 ml to each
Source plate 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.
LB Agar
3. Incubate the vial of cells for 30 - 60 minutes in a 37°C incuba-
tion oven.
Incubate Source plates
Growth should be evident (Broth should be slightly turbid or
overnight at 37°C
cloudy). If growth is not evident, incubate for a longer period
of time.
Source Plate
E. coli Cells 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).
Students begin 5. With the same loop, streak through the

Experiment cells once or twice into another clean
Protocol B section of the plate (figure bottom
right) to obtain isolated colonies.
Students
+DNA
transfer 2-3
large colonies 6. Label the plates "E. coli", invert and incubate the plates
-DNA
overnight (16-24 hours) at 37°C in an incubation oven.

250 µl
CaCl2
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.
Pre-Lab Preparations - Protocol B
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. Using an automatic micropipet, dispense 12 µl of the supercoiled

pBR322 DNA to each of 10 microtest tubes labeled "pBR322 DNA".
10. Cap the tubes and place them on ice.
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.
Recovery Broth
Summary of Reagent Preparations Dispense recovery broth (optional) or set up

a classroom pipeting station.
5 plates E. coli cells
20 plates NO AMP (No Stripe) 5 ml 12. Dispense 1.5 ml Recovery Broth into 10
20 plates PLUS AMP (Stripe) 5 ml sterile tubes labeled "Recovery Broth"
using a sterile pipet.
10 tubes pBR322 DNA 25 µl on ice
10 tubes Control Buffer 25 µl on ice 13. Cap the tubes and place them in the re-
10 tubes Recovery Broth (optional) 1.5 ml 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)
Protocol Hint and Optional Activity:
Do not discard the tubes containing transformed bacteria. After plating

an aliquot on selection plates, add an additional 50 µl of recovery broth
to 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. Remove all except 50 µl of medium (supernatant, top layer).
3. Resuspend the cell pellet in remaining medium.
4. Spread the entire contents of the tube on selection medium.
5. Incubate the plate as before, 16-24 hours in a 37°C incubation oven.
6. Follow proper procedures for disposal of contaminated materials.
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.
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.
• The control plate that is labeled “PLUS AMP Control” shows
that cells without the plasmid will not grow in the presence of
ampicillin.
• The control plate labeled “NO AMP Control” shows that the
cells without the plasmid are able to grow on agar without
ampicillin.
• The plate “NO AMP pBR322 DNA” shows that the cells were not
damaged during the transformation process and therefore are
able to grow on agar plates that do not contain ampicillin.
Idealized Schematic of Results - Protocol A and B


White colonies No colonies White colonies White colonies

NO AMP plated Plus AMP plated NO AMP plated Plus AMP plated
with control cells with control cells with cells with cells
- pBR322 DNA - pBR322 DNA + pBR322 DNA + pBR322 DNA
Result: white colo- Result: No growth Result: Both trans- Result: individual
nies. May look like a formed and untrans- white colonies.
smeared layer of cells Demonstrates: Cells are formed cells will
referred to as a "lawn". sensitive to ampicillin. grow as white colo- Demonstrates: Only
Without pBR322 DNA nies. May look like a transformed cells
Demonstrates: Original they are not ampicillin- smeared layer of cells. which are resistant to
untransformed cells are resistant. ampicillin due to the
viable in the absence of Demonstrates: Trans- uptake of pBR322 DNA
ampicillin. formed cells are viable will grow.
in the absence of
ampicillin.
Study Questions and Answers
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.
Section V - Reactivity Data
Material Safety Data Sheet Stability Unstable Conditions to Avoid
May be used to comply with OSHA's Hazard Communication
EDVOTEK ® Standard. 29 CFR 1910.1200 Standard must be consulted for Stable X Incompatibles
specific requirements. Incompatibility Strong oxidizers
Hazardous Decomposition or Byproducts Toxic oxides of carbon, nitrogen and sulfur

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
Ampicillin be marked to indicate that.
Hazardous May Occur Conditions to Avoid
Section I Polymerization Will Not Occur X Incompaticles
Manufacturer's Name Emergency Telephone Number
(301) 251-5990 Section VI - Health Hazard Data
EDVOTEK, Inc. Route(s) of Entry: Inhalation? Skin? Ingestion? Yes
Telephone Number for information Yes Yes
Address (Number, Street, City, State, Zip Code) (301) 251-5990
Health Hazards (Acute and Chronic)
Date Prepared Sensitizers may result in allergic reaction
14676 Rothgeb Drive 07/01/03
Carcinogenicity: No data NTP? IARC Monographs? OSHA Regulation?
Rockville, MD 20850 Signature of Preparer (optional)
Signs and Symptoms of Exposure Repeated exposure may result in sensitization and possible
anaphylactic shock.
Section II - Hazardous Ingredients/Identify Information
Medical Conditions Generally Aggravated by Exposure No data
Hazardous Components [Specific Other Limits
Chemical Identity; Common Name(s)] OSHA PEL ACGIH TLV Recommended % (Optional)
Ampicillin Emergency First Aid Procedures Ingestion: Allergic symptoms.
CAS# 7177-48-2 No data Eyes/Skin: Flush with water Inhalation: Move to fresh air
Section VII - Precautions for Safe Handling and Use

Section III - Physical/Chemical Characteristics Steps to be Taken in case Material is Released for Spilled Wear suitable protective clothing. Sweep up
Boiling Point No data Specific Gravity (H 0 = 1) No data and place in suitable container for later disposal. Do not flush spilled material down sink.
2
Vapor Pressure (mm Hg.) No data Melting Point No data Waste Disposal Method Observe all federal, state, and local regulations
No data Evaporation Rate No data

Vapor Density (AIR = 1)
(Butyl Acetate = 1) Precautions to be Taken in Handling and Storing
Solubility in Water Keep away from incompatible substances
Slightly soluble
Appearance and Odor Other Precautions None

Odorless, white crystaline powder
Section IV - Physical/Chemical Characteristics N.D. = No data Section VIII - Control Measures

Flash Point (Method Used) Flammable Limits LEL UEL
Respiratory Protection (Specify Type)
No data N.D. N.D.
Extinguishing Media Ventilation Local Exhaust Yes Special None
Dry chemical, carbon dioxide, water spray or regular foam
Mechanical (General) No Other None
Special Fire Fighting Procedures
Move container from fire area if possible. Do not scatter spilled Protective Gloves Yes Eye Protection Splash or dust proof
material with water streams.
Other Protective Clothing or Equipment Eye wash
Unusual Fire and Explosion Hazards
Avoid breathing vapors. Work/Hygienic Practices Wear protective clothing and equipment to prevent contact.
Section V - Reactivity Data

Material Safety Data Sheet Stability Unstable Conditions to Avoid
May be used to comply with OSHA's Hazard Communication
EDVOTEK ® Standard. 29 CFR 1910.1200 Standard must be consulted for
specific requirements.
Stable X Avoid incompatibles
Incompatibility
Strong oxdizers
IDENTITY (As Used on Label and List) Note: Blank spaces are not permitted. If any item is not Hazardous Decomposition or Byproducts
applicable, or no information is available, the space must Sulfur dioxide, mercaptans, carbon monoxide, carbon dioxide, formaldehyde
Solvent for Induction of Competency be marked to indicate that.
Hazardous May Occur Conditions to Avoid
Section I Polymerization X
Will Not Occur
Manufacturer's Name Emergency Telephone Number
(301) 251-5990 Section VI - Health Hazard Data
EDVOTEK, Inc. Route(s) of Entry: Inhalation? Skin? Ingestion?
Telephone Number for information
Address (Number, Street, City, State, Zip Code) Yes Yes Yes
(301) 251-5990
Health Hazards (Acute and Chronic) Inhalation/Ingestion: Nausea and vomiting
Date Prepared
14676 Rothgeb Drive 05-25-05 Skin/eye contact: Rapid absorption causing irritation
Rockville, MD 20850 Carcinogenicity: NTP? IARC Monographs? OSHA Regulation?
Signature of Preparer (optional) None identified
Signs and Symptoms of Exposure
Section II - Hazardous Ingredients/Identify Information Irritation
Hazardous Components [Specific Other Limits Medical Conditions Generally Aggravated by Exposure
Chemical Identity; Common Name(s)] OSHA PEL ACGIH TLV Recommended % (Optional) Skin disorders
None ------------------- Not established ------------------ Emergency First Aid Procedures
Ingestion: Call medical help, do not induce vomiting Inhalation: remove to fresh air
Skin/eye contact: Flush w/ water
Section VII - Precautions for Safe Handling and Use
Section III - Physical/Chemical Characteristics
Steps to be Taken in case Material is Released for Spilled
Boiling Point Specific Gravity (H 0 = 1) Wear protective clothing. Take up with sand or other absorbant and place in container
No data 2 No data
Dispose of properly.
Vapor Pressure (mm Hg.) Melting Point Waste Disposal Method
No data No data Observe all federal, state, and local regulations.
Evaporation Rate
Vapor Density (AIR = 1)
No data (Butyl Acetate = 1) No data Precautions to be Taken in Handling and Storing
Solubility in Water
Soluble Avoid contact
Appearance and Odor Other Precautions
Clear liquid None
Section IV - Physical/Chemical Characteristics

Section VIII - Control Measures
Flash Point (Method Used) Flammable Limits LEL UEL
No data No data No data Respiratory Protection (Specify Type) SCBA
Extinguishing Media Local Exhaust Special
Use water spray, alcohol foam, dry chemical, or carbon dioxide Ventilation Yes None
Mechanical (General) Yes Other None
Special Fire Fighting Procedures
Wear protective equipment and SCBA with full facepiece. Move container from fire Protective Gloves Butyl rubber gloves Eye Protection Safety goggles
area if possible.
Other Protective Clothing or Equipment
Unusual Fire and Explosion Hazards Uniform or apron
Vapors may flow along surfaces and flash back. Work/Hygienic Practices
Avoid contact

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The Biotechnology Education Company®

Storage: See Page 3 for

No IPTG used in this experiment.

All components are intended for educational research only.

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

Protocol B (AP Biology) 15

Experiment Results and Analysis

Determination of Transformation Efficiency 22

Notes to the Instructor 25

Pour Agar Plates 27

Experiment Results and Analysis

Idealized Schematic of Results 36

Study Questions and Answers 37

Material Safety Data Sheets 38

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

Important READ ME!

This transformation experiment can be conducted by two different methods.

Protocol A: Conventional Method Using EDVOTEK

Protocol B: Alternate Method (1997) for Advanced

Instructor’ Pre-lab Prep

Plate cells and grow

After choosing a protocol option, perform all pre-lab and experimental

EDVOTEK - The Biotechnology Education Company ®

Storage: Store components listed below at Room temperature

• Petri plates, small

All components are

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

• Automatic Micropipet (5-50 µl) and tips

* If a second water bath is not available, water can be heated to 42°C in

Visit our web site for information

1-800-EDVOTEK FAX: (301) 340-0582

• The experiment number and title

EDVOTEK - The Biotechnology Education Company ®

Bacterial transformation is of central importance in molecular biology. It

DNA was the genetic material.

The transformation process involves the

Much of the current research and experimentation in molecular biology

The amount of cells transformed per 1 microgram of DNA is called the

After the incubation of plates with the transformed cells, colonies

grew from one transformed cell, the transformation efficiency can be

Plasmid DNAs are extrachromosomal, double-stranded circular molecules

In bacterial cells, plasmids are present primarily in supercoiled forms.

Before you start the experiment

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

Brief Description of Experiment:

This transformation experiment can be conducted by two different

Protocol A: Conventional Method Using EDVOTEK

Protocol B: Alternate Method (1997) for Advanced

Important READ ME!

Transformation experiments contain antibiotics which are used for the

2. Exercise extreme caution when working with equipment which is

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

4. The E. coli bacteria used in this experiment is not considered patho-

5. Properly dispose materials after completing the experiment:

B. All materials, including petri plates, pipets, transfer pipets,

• Autoclave at 121° C for 20 minutes.

• Soak in 10% bleach solution.

Protocol A - Conventional Method Using EDVOTEK Lyphocells™

Transformation Experiment Flow Chart

Add 0.75 ml recovery broth

For optimal results, store

NO AMP PLUS AMP NO AMP PLUS AMP