The Biotechnology Education Company®
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sed
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ated
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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 bacterial 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.
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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
Pre-Lab Preparations
Pour Agar Plates 27
Protocol A
30
Protocol B
32
Protocol Hint and Optional Activity
Experiment Results and Analysis
34
Experiment Questions
34
Idealized Schematic of Results
36
Study Questions and Answers
Material Safety Data Sheets
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37
38
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EDVO-Kit # 201
Transformation of E. coli with pBR322
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 B
AP Biology
Lab 6A
Protocol A
Conventional
Method
Resuspended
Freeze-dried
E. coli cells
Plate cells and grow
overnight at 37°C
Add Competency
Induction Solvent
and Incubate on ice
for 30 minutes
Students
transfer 2-3
large colonies
Students
transfer
300 µl Cells
pBR322
DNA
250 µl
CaCl2
Control
Buffer
Students begin
Experiment
Protocol A
-DNA
Students begin
Experiment
Protocol B
Aliquot for
each group
0.7 ml
Cell Suspension
+DNA
cells
Instructor’ Pre-lab Prep
Incubate vial
of cells
overnight at 37°C
Instructor’ Pre-lab Prep
There are subtle differences between the two
protocols provided with
this experiment. The
major difference between the two options
are illustrated in the
figure at right.
After choosing a protocol option, perform all pre-lab and experimental
procedures consistently in accordance with instructions for the chosen
protocol.
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EDVO-Kit # 201
Transformation of E. coli with pBR322
Experiment Components
Storage: Store components A-G in the refrigerator.
Component
Quantities:
Experiment # 201 is
designed for 10 groups
(2-4 students per
group).
A
B
C
D
E
F
G
Transformation LyphoCells™ (DO NOT FREEZE)
Supercoiled pBR322 DNA
Control Buffer (no DNA) (not used in Protocol B)
Ampicillin
Cell reconstitution medium
Solvent for induction of competency (not used in Protocol B)
CaCl2 (not used in Protocol A)
Storage: Store components listed below at Room temperature
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.
•
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
•
•
•
•
•
•
•
Important READ ME!
In this experiment, antibiotics are used for the selection of transformed bacteria. Students who have or suspect to have allergies to penicilllin, ampicillin or tetracycine should not participate in this experiment.
None of the experiment
components are derived
from human sources.
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Transformation of E. coli with pBR322
EDVO-Kit # 201
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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Please have the following information:
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• The literature version number
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6
EDVO-Kit # 201
Transformation of E. coli with pBR322
Background Information
Bacterial Transformation
Bacterial transformation is of central importance in molecular biology. It
allows for the propagation of recombinant DNA molecules that are constructed in vitro or found in nature. Transformation is also of historical
importance since it led to the discovery by Avery, in the late 1940’s, that
DNA was the genetic material.
final vol at
Number of
Number of
transformants X recovery (ml) =
transformants
vol plated (ml)
µg of DNA
per µg
Specific example:
100
transformants
0.01 µg
X
1 ml
0.1 ml
=
100,000
(1 x 105)
transformants
per µg
Figure 1:
Bacterial Transformation Efficiency Calculation
The transformation process involves the
uptake of exogenous DNA by the cell
which results in a newly acquired genetic
trait that is stable and heritable. Bacterial cells must be in a particular physiological state before they can be transformed.
This state is referred to as competency.
Competency can occur naturally in certain species of Haemophilus and Bacillus
when the levels of nutrients and oxygen are low. Competent Haemophilus
express a membrane associated transport complex which binds and transfers
certain DNA molecules from the medium
into the cell where they are incorporated
and their genes are expressed. In nature,
the source of external DNA is from other
cells that have died and lysed.
Much of the current research and experimentation in molecular biology
involves the transformation of E. coli which does not enter a stage of
natural competency. E. coli can be artificially induced to enter competency 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. Competent 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 antibiotics 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/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 © 1987, 88, 89, 90, 93, 94, 95, 97, 98, 99, 2005, 2009 EDVOTEK, Inc., all
rights reserved
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Transformation of E. coli with pBR322
EDVO-Kit # 201
7
Bacterial Transformation
Plasmid DNAs are extrachromosomal, double-stranded circular molecules
that are found in various strains of bacteria. Many plasmids contain
genes that code for antibiotic resistance. E. coli plasmid pBR322
consists of 4,362 base pairs and contains antibiotic resistance genes
for ampicillin (Amp) and tetracycline (Tet). Ampicillin is a derivative
of penicillin and inhibits bacterial growth by interfering with the
synthesis of cell walls. The product of the ampicillin resistance gene
is beta-lactamase. This enzyme is secreted by transformed bacterial
cells and destroys the ampicillin in the surrounding agar medium.
Subsequently, cells that were not transformed are able to undergo
limited growth in the zones that have been cleared of ampicilRelaxed
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 loncircular 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.
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 © 1987, 88, 89, 90, 93, 94, 95, 97, 98, 99, 2005, 2009 EDVOTEK, Inc., all
rights reserved
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Background Information
grew from one transformed cell, the transformation efficiency can be
calculated. For example, if 100 colonies are present, the transformation efficiency can be calculated as outlined in Figure 1. In research
laboratories, bacterial transformation efficiencies generally range from
1 x 106 to 1 x 108 cells per microgram of DNA. There are procedures
which can produce cells having transformation efficiencies approaching
1010. Transformation is never 100%; in a typical experiment, 1 in 10,000
cells successfully incorporate the transforming plasmid. However, there
is such a large number of bacterial host cells in a sample (typically 1 x
109) and only a small fraction of cells need to be transformed to obtain
colonies on a plate. Transformation efficiency can be demonstrated by
plating equal volumes (0.1 ml) of recovered cells on selective (containing 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.
8
EDVO-Kit # 201
Transformation of E. coli with pBR322
Experiment Overview
Before you start the experiment
The Experiment
1. Read all instructions before starting the experiment.
2. Write a hypothesis that reflects the experiment and predict experimental outcomes.
Experiment Objective:
The objective of this experiment is to develop an understanding of
bacterial transformation by plasmid DNA. This experiment enables the
students to observe an acquired phenotypic trait exhibited by transformed 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.
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 © 1987, 88, 89, 90, 93, 94, 95, 97, 98, 99, 2005, 2009 EDVOTEK, Inc., all
rights reserved
EVT 091102K
The Biotechnology Education Company ® • 1-800-EDVOTEK • www.edvotek.com
Transformation of E. coli with pBR322
EDVO-Kit # 201
9
Laboratory Safety
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.
2. Exercise extreme caution when working with equipment which is
used in conjunction with the heating and/or melting of reagents.
3. Do not mouth pipet reagents - use pipet pumps or bulbs.
4. The E. coli bacteria used in this experiment is not considered pathogenic. Although it is rarely associated with any illness in healthy
individuals, it is good practice to follow simple safety guidelines in
handling and disposal of materials contaminated with bacteria.
5. Properly dispose materials after completing the experiment:
A. Wipe down the lab bench with a 10% bleach solution or a laboratory disinfectant.
B. All materials, including petri plates, pipets, transfer pipets,
loops and tubes, that come in contact with bacteria should be
disinfected 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 prevent 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 materials into a tub containing a 10% bleach solution. Soak the materials overnight and then discard. Wear gloves and goggles
when working with bleach.
6. Wear gloves, and at the end of the experiment, wash hands thoroughly with soap and water.
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 © 1987, 88, 89, 90, 93, 94, 95, 97, 98, 99, 2005, 2009 EDVOTEK, Inc., all
rights reserved
EVT 091102K
The Biotechnology Education Company ® • 1-800-EDVOTEK • www.edvotek.com
The Experiment
1. Gloves and goggles should be worn routinely as good laboratory
practice.
10
EDVO-Kit # 201
Transformation of E. coli with pBR322
Protocol A - Conventional Method Using EDVOTEK Lyphocells™
Transformation Experiment Flow Chart
0.7 ml
Cells
The Experiment
0.25 ml
Cells
0.25 ml
Cells
25 µl
pBR322 DNA
25 µl Control
Buffer
Control
Buffer
pBR322
DNA
Incubate on ice for
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 upright position after streaking
to allow the cell suspension to be absorbed by the
agar. After approximately
20 minutes, invert the plates
for overnight incubation at
37°C.
Streak 0.25 ml of cell
suspension on plates
NO AMP
Control
PLUS AMP
Control
Incubate
overnight in 37°C
incubation oven
NO AMP
Control
PLUS AMP
Control
White Colonies
No Colonies
NO AMP
pBR322 DNA
PLUS AMP
pBR322 DNA
Incubate
overnight in 37°C
incubation oven
NO AMP
pBR322 DNA
PLUS AMP
pBR322 DNA
White Colonies
White Colonies
Plates contaning antibiotic have a stripe on the side of the plate.
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 © 1987, 88, 89, 90, 93, 94, 95, 97, 98, 99, 2005, 2009 EDVOTEK, Inc., all
rights reserved
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Transformation of E. coli with pBR322
EDVO-Kit # 201
11
Protocol A - Conventional Method Using EDVOTEK Lyphocells™
Setting up the Transformation and Control
Experiment
•
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.
•
Cap the tube; mix by tapping. Put
the tube back on ice.
Control
Buffer
pBR322
DNA
Con
Bufftrol
er
pBR
22
DN3
A
2. Set up the Control:
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”.
1 ml
•
Cap the tube; mix by tapping. Put the tube back on ice.
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
42°C for 90 seconds.
This heat shock step facilitates the
entry of DNA in bacterial cells.
+ DNA
~ 0.06 ml
42°C
- DNA
0.25 ml
< 0.02 ml
+ DNA
6. Return both tubes to incubate on ice
for for 1 minute.
- DNA
Guide for Sterile
Calibrated
Transfer Pipet
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 © 1987, 88, 89, 90, 93, 94, 95, 97, 98, 99, 2005, 2009 EDVOTEK, Inc., all
rights reserved
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The Experiment
1. Put your initials or group number on the
tubes labeled “pBR322 DNA” (contains 25 µl
of plasmid DNA) and “Control Buffer” (contains 25 µl of buffer) . Place them back on ice. 12
EDVO-Kit # 201
Transformation of E. coli with pBR322
Protocol A - Conventional Method Using EDVOTEK Lyphocells™
7. Add 0.75 ml of the Recovery Broth to
the tube “Control Buffer”.
8. Add 0.75 ml of the Recovery Broth
to the tube “pBR322 DNA”.
pBR322
DNA
Add the recovery broth with a sterile
1 ml pipet. Avoid touching the
cells with the pipet.
Control
Buffer
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.
+ DNA
- DNA
37°C
9. Incubate the closed tubes in a 37°C water
bath for 30 minutes for a recovery period.
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:
Label one unstriped plate:
NO AMP Control NO AMP pBR322 DNA
•
•
Label one striped plate:
Label one striped plate:
PLUS AMP Control
PLUS AMP pBR322 DNA
•
Put your initials or group number on all the plates.
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.
pBR322
DNA
Control
Buffer
The Experiment
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 © 1987, 88, 89, 90, 93, 94, 95, 97, 98, 99, 2005, 2009 EDVOTEK, Inc., all
rights reserved
EVT 091102K
The Biotechnology Education Company ® • 1-800-EDVOTEK • www.edvotek.com
EDVO-Kit # 201
Transformation of E. coli with pBR322
13
Protocol A - Conventional Method Using EDVOTEK Lyphocells™
Plating the cells
Plating cells from the tube labeled "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
one direction
Same plate spread cells 90°
to first direction
To avoid contamination when plating, do not
set the lid down on the lab bench -- Lift the
lid of the plate only enough to allow spreading. Be careful to avoid gouging the loop
into the agar.
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 • 0.25 ml to the plate labeled
NO AMP pBR322 DNA
PLUS AMP pBR322 DNA
16. Spread the cells with a sterile inoculating loop.
Reminder:
Follow proper
procedures
for disposal of
contaminated
materials.
17. Cover the plate and allow the liquid to be absorbed (approximately
15-20 minutes).
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 © 1987, 88, 89, 90, 93, 94, 95, 97, 98, 99, 2005, 2009 EDVOTEK, Inc., all
rights reserved
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The Experiment
12. Use a fresh, sterile 1 ml pipet to transfer recovered cells from the
tube "Control Buffer" to the middle of the following plates:
• 0.25 ml to the plate labeled NO AMP Control
• 0.25 ml to the plate labeled
PLUS AMP Control
14
EDVO-Kit # 201
Transformation of E. coli with pBR322
Protocol A - Conventional Method Using EDVOTEK Lyphocells™
Preparing Plates for Incubation
The Experiment
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.
If you do not have an incubation oven, the plates can
be left at room temperature. Colonies of transformed 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
medium, it is best to incubate the plates upright.
The plates are inverted to prevent condensation on the lid, which could drip onto the
culture and interfere with experimental results.
Group 4
Reminder:
Follow proper
procedures
for disposal of
contaminated
materials.
20. Place the set of plates in a safe place designated by your instructor.
The plates should be left in the upright position to allow the cell
suspension to be absorbed by the agar for 15 - 20 minutes.
Viewing Plates After Incubation
22. Proceed to analyzing your results.
23. After analyzing your results, follow proper procedures for disposal
of contaminated materials.
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 © 1987, 88, 89, 90, 93, 94, 95, 97, 98, 99, 2005, 2009 EDVOTEK, Inc., all
rights reserved
EVT 091102K
The Biotechnology Education Company ® • 1-800-EDVOTEK • www.edvotek.com
EDVO-Kit # 201
Transformation of E. coli with pBR322
15
Protocol B - Advanced Placement Biology Lab 6A
Transformation Experiment Flow Chart
Prepare Source Plates
DAY BEFORE LAB
* The 37°C incubation for
30 minutes (after adding
the Recovery Broth) is not
described in the AP Biology
Student Laboratory Manual
(1997 Edition D). However,
EDVOTEK recommends
that you perform this step
to allow the cells to recover and begin to express the
antibiotic resistance genes.
The Recovery Broth does
not contain antibiotic.
The Experiment
E.coli cells
+ DNA
- DNA
Transfer 2-3
large colonies
+
10 µl
0.25 ml
CaCl 2
pBR322
DNA
(pAMP)
Incubate on ice
for 15 minutes
Incubate at 42°C
for 90 seconds
37°C for
30 minutes
Streak 0.25 ml of
- DNA cells
on each plate
NO AMP
Control
PLUS AMP
Control
Incubate
overnight in 37°C
incubation oven
NO AMP
Control
Add 0.25 ml
Luria
Recovery Broth
PLUS AMP
Control
Plates contaning
antibiotic have a stripe
on the side of the plate.
+ DNA
- DNA
Incubate on ice
for 2 minutes
Streak 0.25 ml of
+ DNA cells
on each plate
NO AMP
pBR322 DNA
PLUS AMP
pBR322 DNA
Incubate
overnight in 37°C
incubation oven
NO AMP
pBR322 DNA
PLUS AMP
pBR322 DNA
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 © 1987, 88, 89, 90, 93, 94, 95, 97, 98, 99, 2005, 2009 EDVOTEK, Inc., all
rights reserved
EVT 091102K
The Biotechnology Education Company ® • 1-800-EDVOTEK • www.edvotek.com
16
EDVO-Kit # 201
Transformation of E. coli with pBR322
Protocol B - Advanced Placement Biology Lab 6A
Setting up the Transformation and Control
Experiment
Contains
1. Label one microcentrifuge tube “ + DNA “. This will be the transformation tube with plasmid DNA.
2. Label a second microcentrifuge tube “
- DNA “. This will be the experimental
control tube without plasmid DNA.
+ DNA
“-”
Does not contain
plasmid DNA
- DNA
“+” 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.
•
Between your fingers, twist the toothpick vigorously and up
and down in the CaCl2 solution to dislodge and emulsify the
cells.
+
pBR322
10 µl
5. Suspend the cells in both tubes by
tapping or vortexing.
6. Add 10 µl of pBR322 DNA to the
tube labeled “ + DNA “.
Avoid scraping up agar when transferring the cells
from the source plate to the tubes with calcium
chloride solution. It is important that the cells are
resuspended in the calcium chloride solution and is
not left on the toothpick or on the wall of the tube.
This plasmid contains the ampicillin
resistance gene, often referred to
as pAMP.
7. Incubate the two
tubes on ice for
15 minutes.
+ DNA
250 µl
CaCl2
•
- DNA
Transfer 2-4 large
colonies to each
tube containing
ice cold
CaCl2
+ DNA
Source Plate
E. coli Cells
- DNA
The Experiment
Reminder
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 © 1987, 88, 89, 90, 93, 94, 95, 97, 98, 99, 2005, 2009 EDVOTEK, Inc., all
rights reserved
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Transformation of E. coli with pBR322
EDVO-Kit # 201
17
Protocol B - Advanced Placement Biology Lab 6A
- DNA
+ DNA
- DNA
42°C
9. Return both tubes immediately to the ice
bucket and incubate for
two minutes.
+ DNA
11. Incubate the cells for 30 minutes in a 37°C waterbath for a
recovery period.*
37°C
+ DNA
* This step (#11) is not described in the
AP Biology Student Laboratory Manual
(1997 Edition D). However, EDVOTEK
recommends that you perform this step
to allow the cells to recover and begin to
express the antibiotic resistance genes.
The Recovery Broth does not contain
antibiotic.
- DNA
- DNA
10. Using a sterile pipet, add 250 µl (0.25 ml) of Recovery Broth to
each tube and mix.
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:
Label one unstriped plate:
NO AMP Control NO AMP pBR322 DNA
•
•
Label one striped plate:
Label one striped plate:
PLUS AMP Control
PLUS AMP pBR322 DNA
•
Put your initials or group number on all the plates.
13. After the recovery period, remove the tubes
from the water bath and place them on
the lab bench. Proceed to plating the
cells for incubation.
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 © 1987, 88, 89, 90, 93, 94, 95, 97, 98, 99, 2005, 2009 EDVOTEK, Inc., all
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+ DNA
•
•
- DNA
The Experiment
+ DNA
8. Place both tubes in a waterbath at 42°C
for 90 seconds for the heat shock step.
This facilitates the entry of DNA in bacterial cells.
18
EDVO-Kit # 201
Transformation of E. coli with pBR322
Protocol B - Advanced Placement Biology Lab 6A
Plating the cells
Plating cells from the tube labeled " - DNA "
Contains
14. 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 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.
The Experiment
Reminder
“+” plasmid DNA
“-”
To avoid contamination when plating, do
not set the lid down
on the lab bench - lift
the lid of the plate
only enough to allow
spreading. Be careful
to avoid gouging the
loop into the agar.
Same plate:
Spread cells
spread cells 90° to
in one direction
first direction
16. Cover both plates and allow the liquid to be absorbed (approximately 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).
.
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 © 1987, 88, 89, 90, 93, 94, 95, 97, 98, 99, 2005, 2009 EDVOTEK, Inc., all
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Transformation of E. coli with pBR322
EDVO-Kit # 201
19
Protocol B - Advanced Placement Biology Lab 6A
Preparing Plates for Incubation
18. Stack your group's set of plates on top of one another and tape
them together.
If you do not have an incubation oven, the plates can
be left at room temperature. Colonies of transformed 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
medium, it is best to incubate the plates upright.
The plates are inverted to prevent condensation
on the lid, which could drip onto the culture and
may interfere with experimental results.
Group 4
Reminder:
Follow proper
procedures
for disposal of
contaminated
materials.
20. Place the set of plates in a safe place designated by your instructor.
The plates should be left in the upright position to allow the cell
suspension to be absorbed by the agar for 15 - 20 minutes.
Viewing Plates After Incubation
22. Proceed to analyzing your results.
23. After analyzing your results, follow proper procedures for disposal
of contaminated materials.
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 © 1987, 88, 89, 90, 93, 94, 95, 97, 98, 99, 2005, 2009 EDVOTEK, Inc., all
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The Experiment
19. Put your initials or group number on the taped set of plates.
20
EDVO-Kit # 201
Transformation of E. coli with pBR322
Experiment Results and Analysis
Laboratory notebook recordings:
The Experiment
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.
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 bacterial 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
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
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Transformation of E. coli with pBR322
EDVO-Kit # 201
21
Experiment Results and Analysis
Data Collection
1. Observe the results you obtained on your transformation and control plates.
Transformation Plate: + DNA
•
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 transformation efficiency values?
If you did not get any results, what factors could be attributed
to this fact?
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 © 1987, 88, 89, 90, 93, 94, 95, 97, 98, 99, 2005, 2009 EDVOTEK, Inc., all
rights reserved
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The Experiment
22
EDVO-Kit # 201
Transformation of E. coli with pBR322
Experiment Results and Analysis
Determination of Transformation Efficiency
The Experiment
Transformation efficiency is a quantitative determination of how many
cells were transformed per 1 µg of plasmid DNA. In essence, it is an indicator of how well the transformation experiment worked.
You will calculate the transformation efficiency from the data you collect 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
transformants
µg of DNA
x
Example: Assume you observed 40 colonies:
40
transformants
0.025 µg
x
1.0 ml
0.25 ml
=
6400
(6.4 x 103)
transformants
per µg
final vol at
recovery (ml)
vol plated (ml)
=
Number of
transformants
per µg
Quick Reference for
Expt. 201 - Protocol A:
25 ng (0.025 µg) of DNA is used.
The final volume at recovery is 1.0 ml.
The volume plated is 0.25 ml.
Quick Reference for
Expt. 201 - Protocol B:
10 ng (0.01 µg) of DNA is used.
The final volume at recovery is 0.50 ml.
The volume plated is 0.25 ml.
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 © 1987, 88, 89, 90, 93, 94, 95, 97, 98, 99, 2005, 2009 EDVOTEK, Inc., all
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Transformation of E. coli with pBR322
EDVO-Kit # 201
23
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?
3. What evidence do you have that transformation was successful?
4. What are some reasons why transformation may not be successful?
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 © 1987, 88, 89, 90, 93, 94, 95, 97, 98, 99, 2005, 2009 EDVOTEK, Inc., all
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The Experiment
2. Why did the recovery broth not contain ampicillin?
24
EDVO-Kit # 201
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Transformation of E. coli with pBR322
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Transformation of E. coli with pBR322
EDVO-Kit # 201
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 antibiotics such as penicilllin, ampicillin, kanamycin or tetracycine should
not participate in this experiment.
Organizing and Implementing the Experiment
Class size, length of laboratory sessions, and availability of equipment
are factors which must be considered in the planning and the implementation of this experiment with your students.
Online Ordering
now available
The guidelines that are presented in this manual are based on ten laboratory groups consisting of two, or up to four students. The following
are implementation guidelines, which can be adapted to fit your specific
set of circumstances. If you do not find the answers to your questions
in this section, a variety of resources are available at the EDVOTEK web
site. In addition, Technical Service is available from 9:00 am to 6:00 pm,
Eastern time zone. Call 1-800-EDVOTEK for help from our knowledgeable technical staff.
Visit our web site for
information about
EDVOTEK's complete
line of experiments for
biotechnology and
biology education.
Day 1: (Prior to the Lab)
Technical Service
Department
E
O
DV
-TE
C H S E RV I C E
1-800-EDVOTEK
(1-800-338-6835)
FAX:
(301) 340-0582
web: www.edvotek.com
ET
Mo
Mon - Fri
9:00 am to 6:00 pm ET
email:
edvotek@aol.com
m
6p
Please have the following information:
n - Fri 9 am
• The experiment number and title
• Kit Lot number on box or tube
• The literature version number
(in lower right corner)
• Approximate purchase date
•
•
•
Prepare agar plates
Prepare E. coli Cells (overnight incubation).
Dispense the DNA and control buffer
Day 2: (Day of Lab Experiment)
•
•
Equilibrate water baths at 37°C and
42°C; incubation oven at 37°C
Students transform cells and plate for
overnight incubation.
Day 3: (Day after Lab Experiment)
•
•
•
Students observe transformants and
controls
Students calculate transformation efficiency
Follow clean up and disposal procedures
as outlined in the Laboratory Safety section.
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25
26
EDVO-Kit # 201
Transformation of E. coli with pBR322
Notes to the Instructor:
National content and skill standards
Instructor’s Guide
By performing this experiment, students will develop skills necessary to
do scientific inquiry, learn new techniques using several types of biotechnology equipment, and will learn standard procedures used in transformation. Analysis of the experiments will provide students the means to
transform an abstract concept into a concrete explanation.
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:
•
LABORATORY
NOTEBOOKS
It is highly recommended
that students maintain a
laboratory notebook to
formulate hypotheses and
to record experimental
procedures and results.
•
•
EDVOTEK Cat. # 1401,
Laboratory DataBook
is recommended.
Guidelines for keeping
a laboratory notebook
is available at the
EDVOTEK web site.
Reconstitute the LyphoCells™, plate for individual colonies, and
incubate at 37°C for 16 to 24 hours before the laboratory (overnight
incubation).
For BOTH Protocols A and B:
1. The agar plates can be prepared several days in advance and
stored inverted (agar side on top) in the refrigerator. Preparation requires approximately 1 hour.
2. Dispensing the DNA and reagents requires approximately 30
minutes. This can be done the day before the lab and stored in
the refrigerator.
3. Allow ample time for the equilibration of the water baths at
37°C and 42°C and a bacterial incubation oven at 37°C on the
day of the experiment.
4. In the experiment, each group will perform the transformation
experiment and plate four sets of bacterial cells. These procedures 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.
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 © 1987, 88, 89, 90, 93, 94, 95, 97, 98, 99, 2005, 2009 EDVOTEK, Inc., all
rights reserved
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Transformation of E. coli with pBR322
EDVO-Kit # 201
27
Pre-Lab Preparations
Pour Agar Plates
(Prior to the Lab experiment)
For optimal results, prepare plates two days prior to plating and set
aside the plates inverted at room temperature.
•
If they are poured more than two days before use, they should be
stored inverted in the refrigerator. Remove the plates from the
refrigerator and store inverted for two days at room temperature
before use.
Wear Hot Gloves
and Goggles during all
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 outlined below. When completely melted, the amber-colored solution
should appear free of small particles.
A. Microwave method:
•
•
•
Heat the bottle on High for two 30 second intervals.
Using a hot glove, swirl and heat on High for an additional
25 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, the bottle will be warm to the touch but not burning hot.
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 © 1987, 88, 89, 90, 93, 94, 95, 97, 98, 99, 2005, 2009 EDVOTEK, Inc., all
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60˚C
Instructor’s Guide
•
28
EDVO-Kit # 201
Transformation of E. coli with pBR322
Pre-Lab Preparations
Label (“Stripe”) the Plates
Instructor’s Guide
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 medium 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.
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 © 1987, 88, 89, 90, 93, 94, 95, 97, 98, 99, 2005, 2009 EDVOTEK, Inc., all
rights reserved
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Transformation of E. coli with pBR322
EDVO-Kit # 201
29
Pre-Lab Preparations
9. Add the entire amount of ampicillin powder to the remaining molten ReadyPour medium in the bottle.
10. Recap the bottle and swirl to completely dissolve the ampicillin.
11. Pour 20 transformation plates
(with ampicillin, striped plates):
Use a fresh 10 ml pipet to pour the twenty (20) striped plates, 5 ml
each.
12. Allow the agar to cool and resolidify.
Reminder:
Follow proper
procedures
for disposal of
contaminated
materials.
Note: If plates will be used within two days, store in a sealable plastic
bag so the plates will not dry out. Store at room temperature, inverted.
If you have extra sterile petri plates on hand, use any remaining medium to pour additional plates for the optional activity described on
page 34.
Summary of Poured Plates
Protocol A
20 control plates - small no stripe
plates: 5 ml each - ReadyPour
medium (no ampicillin)
20 transformation plates - small striped
plates: 5 ml each - ReadyPour
medium with ampicillin
Summary of Poured Plates
Protocol B
5 source plates - large plates:
10 ml each - ReadyPour medium
20 control plates - small no stripe
plates: 5 ml each - ReadyPour
medium (no ampicillin)
20 transformation plates - small striped
plates: 5 ml each - ReadyPour
medium with ampicillin
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 © 1987, 88, 89, 90, 93, 94, 95, 97, 98, 99, 2005, 2009 EDVOTEK, Inc., all
rights reserved
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Instructor’s Guide
Add reagents to medium which has been
cooled. Hot medium
will cause reagents
such as ampicillin to
rapidly decompose.
30
EDVO-Kit # 201
Transformation of E. coli with pBR322
Pre-Lab Preparations - Protocol A
Add 6 ml
reconstitution
media (E) to
LyphoCells
Incubate vial of cells
overnight at 37°C
Preparation of E. coli Cells
1. Use a 10 ml sterile pipet to add 6 ml cell reconstitution medium (E) to the vial of LyphoCells.
2. Replace the rubber stopper and cap. Mix by gently
inverting until the freeze dried plug is dissolved.
3. 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
4. Completely thaw the competency
induction solvent (F) and place on
ice. (If there is a white precipitate
in the bottle, warm it in a 37°C waterbath to dissolve the precipitate.)
Add 3 ml
ice cold
Competency
Induction
Solvent (F)
5. Mix and resuspend the vial of incubated cells by inverting and gently
shaking. Place the vial on ice for 10
minutes.
Incubate on ice
30 minutes
The solvent
for induction
of competency
contains salts
to make cells
competent.
6. Use a 10 ml sterile pipet to add 3 ml of ice cold competency induction solvent (F) to the vial of cells.
Dispense
0.7 ml
Cell
Suspension
Cells
Instructor’s Guide
Day before the experiment
Students begin
Experiment
Protocol A
The competency induction solvent is very viscous.
Make sure that a portion of the solvent is not left on
the walls of the pipet.
7. Mix the cells and induction solvent thoroughly by
inverting the vial several times. The solution should
have no dense layers, "streams" or globules (i.e. it
should be a uniform suspension).
8. Keep the cells on ice for a minimum of 30 minutes.
Cells can be kept on ice for up to 3 hours.
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 © 1987, 88, 89, 90, 93, 94, 95, 97, 98, 99, 2005, 2009 EDVOTEK, Inc., all
rights reserved
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Transformation of E. coli with pBR322
EDVO-Kit # 201
31
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.
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.
Summary of Reagent Preparations
20 plates NO AMP (No Stripe)
20 plates PLUS AMP (Stripe)
10 tubes pBR322 DNA
10 tubes Control Buffer
10 tubes Cells 10 tubes Recovery Broth
5 ml
5 ml
25 µl on ice
25 µl on ice
0.7 ml on ice
1.5 ml
16. Using an automatic micropipet, dispense 25 µl of the supercoiled pBR322
DNA to each of 10 microtest tubes
labeled "pBR322 DNA".
17. Cap the tubes and place them on ice.
18. Using a FRESH micropipet tip, dispense
25 µl of control buffer to each of 10 microtest tubes labeled "Control Buffer".
19. Cap the tubes and place them on ice.
Each Group Requires:
Recovery Broth
1
1
1
2
2
4
2
1
Set up a classroom pipeting station, or dispense recovery broth (optional).
tube pBR322 DNA
25 µl on ice
tube Cells
0.7 ml on ice
tube Control Buffer
25 µl on ice
striped plates
unstriped plates
sterile 1 ml pipets
sterile inoculating loops
tube Recovery broth (optional) 1.5 ml
20. Dispense 1.5 ml Recovery Broth into 10
sterile tubes labeled "Recovery Broth"
using a sterile pipet.
21. Cap the tubes and place them in the refrigerator if not to be used immediately.
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 © 1987, 88, 89, 90, 93, 94, 95, 97, 98, 99, 2005, 2009 EDVOTEK, Inc., all
rights reserved
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Instructor’s Guide
12. Use a sterile pipet to aliquot 0.7 ml of cells to 10 ice cold tubes
labeled "Cells"
32
EDVO-Kit # 201
Transformation of E. coli with pBR322
Pre-Lab Preparations - Protocol B
Add 6 ml
reconstitution
media (E) to
LyphoCells vial
Incubate cells
30 - 60 minutes at 37°C
This experiment requires preparation of isolated E.coli host
transformation colonies 16-24 hours before the laboratory experiment, so plan accordingly.
Important: Do not prepare source plates more than 24 hours
before the experiment. Old source plates will compromise the
success of the transformation experiment.
Preparation of E. coli Cells
Transfer 0.3 ml to each
Source plate
LB Agar
1. Use a 10 ml sterile pipet to add 6 ml of cell reconstitution
medium (E) to the vial of LyphoCells.
2. Replace the rubber stopper and cap. Mix by gently inverting
until the freeze dried plug is dissolved.
3. Incubate the vial of cells for 30 - 60 minutes in a 37°C incubation oven.
Incubate Source plates
overnight at 37°C
Source Plate
E. coli Cells
Students
transfer 2-3
large colonies
250 µl
CaCl2
-DNA
Students begin
Experiment
Protocol B
+DNA
Instructor’s Guide
Day before the experiment
Growth should be evident (Broth should be slightly turbid or
cloudy). If growth is not evident, incubate for a longer period
of time.
4. Using a sterile loop, dip into the cell
suspension to remove a loop full of
bacterial. Plate the inoculum on one
quadrant of the plate with the loop
(figure top right).
5. With the same loop, streak through the
cells once or twice into another clean
section of the plate (figure bottom
right) to obtain isolated colonies.
6. Label the plates "E. coli", invert and incubate the plates
overnight (16-24 hours) at 37°C in an incubation oven. 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.
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 © 1987, 88, 89, 90, 93, 94, 95, 97, 98, 99, 2005, 2009 EDVOTEK, Inc., all
rights reserved
EVT 091102K
The Biotechnology Education Company ® • 1-800-EDVOTEK • www.edvotek.com
Transformation of E. coli with pBR322
EDVO-Kit # 201
33
Pre-Lab Preparations - Protocol B
Day of the experiment
Preparation of DNA and Reagents
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
Dispense recovery broth (optional) or set up
a classroom pipeting station.
Summary of Reagent Preparations
5 plates E. coli cells
20 plates NO AMP (No Stripe)
5 ml
20 plates PLUS AMP (Stripe)
5 ml
10 tubes pBR322 DNA
25 µl on ice
10 tubes Control Buffer
25 µl on ice
10 tubes Recovery Broth (optional) 1.5 ml
12. Dispense 1.5 ml Recovery Broth into 10
sterile tubes labeled "Recovery Broth"
using a sterile pipet.
13. Cap the tubes and place them in the refrigerator if not to be used immediately.
Each Group Requires:
•
•
•
•
•
•
•
•
•
•
1
1
1
2
2
2
2
4
2
1
tube (12 µl) pBR322 DNA (on ice)
tube (1 ml) CaCl2 (on ice)
plate E. coli (every 2 groups can share)
empty microcentrifuge tubes
toothpicks
striped plates
unstriped plates
sterile 1 ml pipets
sterile inoculating loops
tube (1.5 ml) Recovery broth (optional)
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 © 1987, 88, 89, 90, 93, 94, 95, 97, 98, 99, 2005, 2009 EDVOTEK, Inc., all
rights reserved
EVT 091102K
The Biotechnology Education Company ® • 1-800-EDVOTEK • www.edvotek.com
Instructor’s Guide
7. Place the tube of supercoiled pBR322 DNA (B) on ice.
34
EDVO-Kit # 201
Transformation of E. coli with pBR322
Protocol Hint and Optional Activity:
Instructor’s Guide
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 microcentrifuge. 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.
Experiment Results and Analysis
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.
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 © 1987, 88, 89, 90, 93, 94, 95, 97, 98, 99, 2005, 2009 EDVOTEK, Inc., all
rights reserved
EVT 091102K
The Biotechnology Education Company ® • 1-800-EDVOTEK • www.edvotek.com
Transformation of E. coli with pBR322
EDVO-Kit # 201
35
Experiment Results and Analysis
3.What is the purpose of the control plates? Explain the difference
between each and why it is necessary to run each.
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 © 1987, 88, 89, 90, 93, 94, 95, 97, 98, 99, 2005, 2009 EDVOTEK, Inc., all
rights reserved
EVT 091102K
The Biotechnology Education Company ® • 1-800-EDVOTEK • www.edvotek.com
Instructor’s Guide
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.
36
EDVO-Kit # 201
Transformation of E. coli with pBR322
Instructor’s Guide
Idealized Schematic of Results - Protocol A and B
NO AMP
Control
PLUS AMP
Control
NO AMP
pBR322 DNA
PLUS AMP
pBR322 DNA
White colonies
No colonies
White colonies
White colonies
NO AMP plated
with cells
+ pBR322 DNA
Plus AMP plated
with cells
+ pBR322 DNA
NO AMP plated
with control cells
- pBR322 DNA
Result: white colonies. May look like a
smeared layer of cells
referred to as a "lawn".
Demonstrates: Original
untransformed cells are
viable in the absence of
ampicillin.
Plus AMP plated
with control cells
- pBR322 DNA
Result: No growth
Demonstrates: Cells are
sensitive to ampicillin.
Without pBR322 DNA
they are not ampicillinresistant.
Result: Both transformed and untransformed cells will
grow as white colonies. May look like a
smeared layer of cells.
Demonstrates: Transformed cells are viable
in the absence of
ampicillin.
Result: individual
white colonies.
Demonstrates: Only
transformed cells
which are resistant to
ampicillin due to the
uptake of pBR322 DNA
will grow.
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 © 1987, 88, 89, 90, 93, 94, 95, 97, 98, 99, 2005, 2009 EDVOTEK, Inc., all
rights reserved
EVT 091102K
The Biotechnology Education Company ® • 1-800-EDVOTEK • www.edvotek.com
Please refer to the kit
insert for the Answers to
Study Questions
EDVOTEK
Section V - Reactivity Data
Material Safety Data Sheet
®
IDENTITY (As Used on Label and List)
Section I
Emergency Telephone Number
Manufacturer's Name
EDVOTEK, Inc.
Telephone Number for information
Address (Number, Street, City, State, Zip Code)
Date Prepared
14676 Rothgeb Drive
Rockville, MD 20850
Incompatibility
(301) 251-5990
(301) 251-5990
07/01/03
Signature of Preparer (optional)
Section II - Hazardous Ingredients/Identify Information
Hazardous Components [Specific
Chemical Identity; Common Name(s)]
OSHA PEL
Ampicillin
CAS# 7177-48-2
ACGIH TLV
Other Limits
Recommended
% (Optional)
Hazardous
Polymerization
No data
Vapor Pressure (mm Hg.)
No data
Melting Point
Vapor Density (AIR = 1)
No data
Evaporation Rate
(Butyl Acetate = 1)
No data
Solubility in Water
Slightly soluble
Appearance and Odor
Odorless, white crystaline powder
Extinguishing Media
N.D. = No data
LEL
N.D.
UEL
Section VI - Health Hazard Data
Route(s) of Entry:
Health Hazards (Acute and Chronic)
Avoid breathing vapors.
Signs and Symptoms of Exposure
Repeated exposure may result in sensitization and possible
anaphylactic shock.
Medical Conditions Generally Aggravated by Exposure
Emergency Telephone Number
Manufacturer's Name
EDVOTEK, Inc.
Address (Number, Street, City, State, Zip Code)
Telephone Number for information
Date Prepared
14676 Rothgeb Drive
Rockville, MD 20850
(301) 251-5990
(301) 251-5990
OSHA PEL
ACGIH TLV
Vapor Pressure (mm Hg.)
No data
Vapor Density (AIR = 1)
No data
Solubility in Water
None
Respiratory Protection (Specify Type)
Protective Gloves
Other Limits
Recommended
% (Optional)
Yes
Eye Protection
Splash or dust proof
Other Protective Clothing or Equipment
Eye wash
Work/Hygienic Practices
Wear protective clothing and equipment to prevent contact.
Unstable
Conditions to Avoid
X
Avoid incompatibles
Strong oxdizers
Hazardous Decomposition or Byproducts
Sulfur dioxide, mercaptans, carbon monoxide, carbon dioxide, formaldehyde
Hazardous
Polymerization
Conditions to Avoid
May Occur
X
Will Not Occur
Section VI - Health Hazard Data
Route(s) of Entry:
Inhalation?
Health Hazards (Acute and Chronic)
Ingestion?
Skin?
Yes
Yes
Yes
Inhalation/Ingestion: Nausea and vomiting
Skin/eye contact: Rapid absorption causing irritation
IARC Monographs?
NTP?
OSHA Regulation?
Irritation
Medical Conditions Generally Aggravated by Exposure
Skin disorders
Emergency First Aid Procedures
Ingestion: Call medical help, do not induce vomiting
Skin/eye contact: Flush w/ water
Inhalation: remove to fresh air
Section VII - Precautions for Safe Handling and Use
Steps to be Taken in case Material is Released for Spilled
No data
Melting Point
No data
Evaporation Rate
(Butyl Acetate = 1)
No data
Wear protective clothing. Take up with sand or other absorbant and place in container
Dispose of properly.
Waste Disposal Method
Observe all federal, state, and local regulations.
Precautions to be Taken in Handling and Storing
Avoid contact
Other Precautions
Clear liquid
Flammable Limits
LEL
No data
UEL
No data
Use water spray, alcohol foam, dry chemical, or carbon dioxide
Special Fire Fighting Procedures
Wear protective equipment and SCBA with full facepiece. Move container from fire
area if possible.
Unusual Fire and Explosion Hazards
None
None
Other
No
Signs and Symptoms of Exposure
Specific Gravity (H 0 = 1)
2
Section IV - Physical/Chemical Characteristics
No data
Special
Yes
Local Exhaust
Soluble
Appearance and Odor
Flash Point (Method Used)
Precautions to be Taken in Handling and Storing
None identified
------------------- Not established ------------------
No data
Observe all federal, state, and local regulations
Carcinogenicity:
Section III - Physical/Chemical Characteristics
Boiling Point
and place in suitable container for later disposal. Do not flush spilled material down sink.
Waste Disposal Method
Incompatibility
05-25-05
Section II - Hazardous Ingredients/Identify Information
None
No data
Ingestion: Allergic symptoms.
Eyes/Skin: Flush with water
Inhalation: Move to fresh air
Stable
Signature of Preparer (optional)
Hazardous Components [Specific
Chemical Identity; Common Name(s)]
OSHA Regulation?
Emergency First Aid Procedures
Stability
Note: Blank spaces are not permitted. If any item is not
applicable, or no information is available, the space must
be marked to indicate that.
Solvent for Induction of Competency
Section I
IARC Monographs?
Section V - Reactivity Data
Material Safety Data Sheet
May be used to comply with OSHA's Hazard Communication
Standard. 29 CFR 1910.1200 Standard must be consulted for
specific requirements.
IDENTITY (As Used on Label and List)
Ingestion? Yes
Yes
NTP?
Mechanical (General)
Move container from fire area if possible. Do not scatter spilled
material with water streams.
®
Skin?
Yes
Sensitizers may result in allergic reaction
Carcinogenicity: No data
Ventilation
Unusual Fire and Explosion Hazards
EDVOTEK
Inhalation?
Incompaticles
Section VIII - Control Measures
N.D.
Dry chemical, carbon dioxide, water spray or regular foam
Special Fire Fighting Procedures
X
Keep away from incompatible substances
Flammable Limits
No data
Conditions to Avoid
May Occur
Will Not Occur
Other Precautions
Section IV - Physical/Chemical Characteristics
Flash Point (Method Used)
Toxic oxides of carbon, nitrogen and sulfur
Steps to be Taken in case Material is Released for Spilled Wear suitable protective clothing. Sweep up
Specific Gravity (H 0 = 1)
2
No data
Extinguishing Media
Hazardous Decomposition or Byproducts
Section VII - Precautions for Safe Handling and Use
Section III - Physical/Chemical Characteristics
No data
Incompatibles
Strong oxidizers
No data
Boiling Point
Conditions to Avoid
X
Stable
Note: Blank spaces are not permitted. If any item is not
applicable, or no information is available, the space must
be marked to indicate that.
Ampicillin
Unstable
Stability
May be used to comply with OSHA's Hazard Communication
Standard. 29 CFR 1910.1200 Standard must be consulted for
specific requirements.
Vapors may flow along surfaces and flash back.
None
Section VIII - Control Measures
Respiratory Protection (Specify Type)
Ventilation
Protective Gloves
Mechanical (General)
Yes
Yes
Butyl rubber gloves
Other Protective Clothing or Equipment
Work/Hygienic Practices
SCBA
Local Exhaust
Uniform or apron
Avoid contact
Special
Other
Eye Protection
None
None
Safety goggles