Bio-Rad Ligation and Transformation Module User Manual
Biotechnology Explorer
™
Ligation and Transformation
Module
Instruction Manual
Catalog #166-5015EDU
explorer.bio-rad.com
For technical support call your local Bio-Rad office or in the U.S. call 1-800-424-6723.
PCR Fragment
This kit is shipped on blue ice. Open immediately upon arrival and store
reagents bags at –20°C.
Duplication of any part of this document is permitted for classroom use only.
Please visit explorer.bio-rad.com to access our selection of language translations
for Biotechnology Explorer kit curricula.
PCR Fragment
Table of Contents
Introduction ..................................................................................1
Kit Inventory Checklist ................................................................6
Safety Issues ................................................................................9
Background ................................................................................10
Quick Guide ................................................................................26
Instructor’s Advance Preparation ............................................32
Student Ligation Protocol..........................................................36
Student Transformation Protocol..............................................40
Appendix A Inoculating a Bacterial Colony for
Plasmid Miniprep ................................................46
Appendix B Restriction Digestion of Plasmid DNA
with Bgl II Enzyme ..............................................48
Introduction
Cloning is the production of multiple exact copies of a piece of DNA, usually
a gene, using molecular biology techniques. Cloning is frequently the first
step of a research project, producing enough DNA for further study.
Using the Ligation and Transformation module students can subclone virtually
any DNA fragment of interest that has been amplified using PCR. We
recommend that the DNA fragment be approximately 200–2,000 base pairs
(bp) in length for best results. Below is a typical workflow for cloning and
sequencing a gene. The steps that the Ligation and Transformation module
enable students to perform are in bold.
The Ligation and Transformation module is part of Bio-Rad’s Cloning and
Sequencing Explorer Series. The Cloning and Sequencing Explorer Series
is a sequence of individual modules that have been designed to work in
concert to give students the real world experience of a molecular biology
research workflow. The additional modules of the Cloning and Sequencing
Explorer Series can be purchased separately. Further information on the
separate modules is available in the Biotechnology Explorer™ catalog or
from explorer.bio-rad.com.
• Amplify gene of interest using PCR
1
• Purify PCR product
2
• Ligation of PCR product into pJet1.2 plasmid
• Transform ligated plasmid into bacteria
• Culture bacteria and grow minipreps
3
• Purify plasmid from minipreps
4
• Analyze plasmid by restriction digestion
• Electrophorese restriction digest reaction
5
• Sequence plasmid and analyze
6
1
GAPDH
PCR module (catalog #166-5010EDU) amplifies a fragment of the
GAPDH
gene
from a preparation of plant genomic DNA.
2
PCR Kleen™ Spin module (catalog #732-6300EDU) purifies 25 PCR products.
3
Microbial Culturing module (catalog #166-5020EDU) contains all required reagents for
culturing bacteria for transformation using the Ligation and Transformation module.
4
Aurum™ Plasmid Mini Purification module (catalog #732-6400EDU) contains reagents to
purify plasmid DNA from 100 minipreps.
5
Electrophoresis modules contain reagents to analyze plasmid restriction digests.
6
Sequencing and Bioinformatics module (catalog #166-5025EDU) is designed to allow
sequencing and bioinformatics analysis of plasmids generated using the Ligation and
Transformation module.
1
Using the Ligation and Transformation module, students will clone a gene
of interest. Prior to starting this laboratory activity, students must have
already amplified a gene of interest using polymerase chain reaction
(PCR) and subsequently purified the PCR product to remove excess
primers, nucleotides, and DNA polymerase, which would otherwise interfere
with subsequent experiments. Students can then use the Ligation and
Transformation module to ligate the DNA fragment into the pJet1.2 blunted
vector, which encodes
amp
r
, an ampicillin-resistance gene. Following ligation,
students will perform transformation to introduce the plasmid into living
bacterial cells.
The pJet1.2 blunted vector enables positive selection of plasmids with the
desired insert due to the disruption of
eco47IR
, an otherwise lethal gene,
that allows growth of successful transformants. Bacteria are then plated
and incubated overnight at 37°C on the selective medium containing
ampicillin and isopropyl b-D-1-thiogalactopyranoside (IPTG), which is
added to increase expression of the
amp
r
gene. Since transformed cells
express an ampicillin-resistance gene, they will grow and divide, each
forming a colony on the plate that is the product of a single transformation
event.
The bacteria containing the cloned gene can be grown in liquid growth
medium and the plasmid containing the insert can be purified from the
bacteria. The pJet1.2 plasmid contains a Bgl II restriction enzyme recognition
site on either side of the insertion site. Using the Bgl II enzyme students
will analyze the cloned plasmid by restriction enzyme digestion and analyze
their digests by agarose gel electrophoresis to confirm the presence of an
insert and determine its size. The resulting fragment can then be compared
to the size of the PCR fragment ligated into the plasmid. Finally, the DNA
fragment can be then sequenced to determine the exact order of
nucleotides in the DNA molecule.
What Skills Do Students Need to Perform this Laboratory
Activity?
This laboratory activity assumes that students and instructors have basic
molecular biology and microbiology skills, such as proper pipeting
techniques, pouring and streaking agar plates and performing agarose gel
2
electrophoresis. In addition, students must understand the principles of
PCR and be able to perform PCR reactions. Bio-Rad’s Biotechnology
Explorer program has a full range of kits to help teach basic skills in individual
laboratories.
What Is the Timeline for Completing the Ligation and
Transformation Protocol?
Before starting this activity, students must have already amplified a gene
of interest using PCR. In addition, the PCR product should be purified to
remove components of the amplification reaction that would otherwise
interfere with the ligation step.
The amount of time it takes to complete the ligation and transformation
protocols depends greatly on the level of your students and whether
additional/optional techniques and analyses are performed in addition to the
basic protocol. Steps using the Ligation and Transformation module are
highlighted in bold. Additionally, there are a few incubation steps that add
to the number of days it takes to complete the laboratory activity. A rough
guide is provided on pages 4 and 5.
3
When Activity to Complete Duration
At least 1 day prior to Run a PCR reaction in thermal 3–4 h
starting the Ligation cycler to amplify a
and Transformation gene of interest
module Electrophorese the PCR 1 h
products (optional)
Purify PCR products 0.5 h
At least 3 days prior to Prepare LB and LB Amp 0.5 h
the transformation step IPTG agar plates
At least 2 days prior to Prepare LB broth 0.5 h
the transformation step Streak
E. coli
on a starter LB 5 min
agar plate
Grow
E. coli
starter plate at 37°C 16+ h
As late as possible the Inoculate starter culture 5 min
day before the Incubate starter culture at
transformation step 37°C in a shaking 8+ h
water bath or incubator
Day of ligation step Ligate PCR product 1 h
Note: Bolded steps use reagents from the Ligation and Transformation
module.
4
When Activity to Complete Duration
Immediately following Transform
E. coli
with ligation 1 h
ligation or during the mixture and plate bacteria on
next laboratory activity LB Amp IPTG agar plates
Incubate transformed bacteria 16+ h
at 37°C
Next day after the Analyze results 0.5 h
transformation step Grow bacterial colony in LB Amp 16+ h
broth for miniprep
Day after growing Perform miniprep plasmid 1 h
bacterial culture for purification to isolate
miniprep plasmid carrying insert
Next laboratory activity Digest plasmid DNA with 1 h
Bgl II restriction enzyme
Analyze digest by agarose gel 1 h
electrophoresis
Prepare the DNA insert for 0.5 h
sequencing
Note: Bolded steps use reagents from the Ligation and Transformation
module.
5
Kit Inventory Checklist
This section lists equipment and reagents necessary to perform the ligation
and transformation protocol in your classroom or teaching laboratory. Each
kit contains sufficient materials for 12 student workstations, 12 ligation
reactions, and 24 transformations. We recommend that students are
teamed up – two to four students per workstation. Please use the checklist
below to confirm inventory.
Kit Components Number/Kit (✔)
T4 DNA ligase, 10 µl 1
❒
Ligation reaction buffer (2x concentration), 100 µl 1 ❒
Proofreading polymerase, 10 µl 1 ❒
pJet1.2 blunted vector, 10 µl 1 ❒
Sterile water, 1 ml 1 ❒
Bgl II restriction enzyme, 50 µl 1 ❒
10x Bgl II reaction buffer, 1 ml 1 ❒
Isopropyl b-D-1-thiogalactopyranoside (IPTG), 1 M, 0.1 ml 1 ❒
Transformation reagent A, 1.25 ml 4 ❒
Transformation reagent B, 1.25 ml 4 ❒
C-growth medium, 30 ml 1 ❒
Microcentrifuge tubes, clear, 1.5 ml 30 ❒
Microcentrifuge tubes, multicolor, 2.0 ml 120 ❒
6
Required Accessories Number/Kit (✔)
PCR product (previously amplified and 1 per team
❒
purified by students)
Microbial Culturing module (catalog #166-5020EDU)* 1
❒
containing the following:
• LB broth capsules (each for making 50 ml of LB broth) 12
❒
• LB nutrient agar powder (to make 500 ml) 1 pouch ❒
• Ampicillin 2 vials ❒
•
E. coli
HB101 K-12, lyophilized bacteria 1 vial ❒
• Culture tubes, sterile, 15 ml 75 ❒
• Petri dishes, sterile 40 ❒
• Sterile inoculating loops 80 ❒
Variable speed microcentrifuge (catalog #166-0602EDU) 1 ❒
Shaking water bath or shaking incubator (37°C) 1 ❒
Water bath (catalog #166-0504EDU), heating block, 1 ❒
(catalog #166-0562EDU)or incubator (70°C)
Adjustable-volume micropipet
0.5–10 µl (catalog #166-0505EDU) 12
❒
20–200 µl (catalog #166-0507EDU) 12 ❒
100–1,000 µl (catalog #166-0508EDU) 12 ❒
Pipet Tips
0.5–10 µl (catalog #223-9354EDU) 1 box
❒
2–200 µl (catalog #223-9347EDU) 1 box ❒
100–1,000 µl (catalog #223-9350EDU) 1 box ❒
Ice bath 1 ❒
Parafilm sealing film 1 ❒
Marking pens 1 ❒
*Note: Standard microbiological reagents may be used in place of the
Microbial Culturing module (see Instructor’s Advanced Prep section for
requirements). Any
E. coli
strain commonly used for transformation (for
example, DH5a, DH10, JM107) may be used in place of
E. coli
HB101.
7
Optional Accessories
Vortex mixer (catalog #166-0610EDU)
Vacuum source
Agarose electrophoresis equipment
GAPDH
PCR module (catalog #166-5010EDU)
PCR Kleen™ Spin Purification module (catalog #732-6300EDU)
pGLO™ Plasmid, 20 µg (catalog #166-0405EDU)
Aurum™ Plasmid Mini Purification Module (catalog #732-6400EDU)
Electrophoresis reagents:
Small Ethidium Bromide DNA Electrophoresis Reagent Pack (catalog
#166-0451EDU)
Small Fast Blast™ DNA Electrophoresis Reagent Pack (catalog
#166-0450EDU)
Sample Loading Dye, 5x, 1 ml (catalog #166-0401EDU)
EZ Load™ 500 bp Molecular Ruler (catalog #170-8354EDU)
Sequencing and Bioinformatics module (catalog #166-5025EDU)
Refills Available Separately
Ligation module reagent refill (catalog #166-5016EDU)
Includes T4 DNA ligase, 2x ligation reaction buffer, proofreading
polymerase, pJet1.2 blunted vector, sterile water
Bgl II reagent refill (catalog #166-5018EDU)
Includes Bgl II restriction enzyme and 10x Bgl II reaction buffer
Transformation module reagent refill (catalog #166-5017EDU)
Includes transformation reagent A, transformation reagent B, 1 M IPTG,
C-growth medium
8
Storage Instructions
The kit is shipped on blue ice. Open immediately upon arrival and store
reagent bags immediately at –20°C.
Safety Issues
Eating, drinking, smoking, and applying cosmetics are not permitted in the
work area. Wearing protective eyewear and gloves is strongly recommended.
Transformation reagent B contains dimethyl sulfoxide (DMSO, CAS #67-68-5),
an organic solvent. Handle with care and follow standard laboratory
practices, including wearing eye protection, gloves, and a laboratory coat
to avoid contact with eyes, skin, and clothing. If the solution comes into
contact with gloves, change the gloves. DMSO passes directly through
latex gloves, readily penetrates skin, and may result in the absorption of
toxic materials and allergens dissolved in the solvent. After handling, wash
hands and any areas that came into contact with the solution thoroughly.
Refer to MSDS for complete safety information.
Ampicillin may cause allergic reactions or irritation to the eyes, respiratory
system, and skin. In case of contact with eyes, rinse immediately with
plenty of water and seek medical advice. Wear suitable protective clothing.
Ampicillin is a member of the penicillin family of antibiotics. Those with
allergies to penicillin or any other member of the penicillin family of
antibiotics should avoid contact with ampicillin.
The
E. coli
HB101 K-12 strain is not pathogenic. However, handling of
E. coli
HB101 K-12 requires the use of standard microbiological practices.
These practices include, but are not limited to, the following: (1) work surfaces
are decontaminated once a day after any spill of viable material; (2) all contaminated liquid or solid wastes are decontaminated before disposal; (3)
persons should wash their hands: (a) after they handle materials involving
organisms containing recombinant DNA molecules, and (b) before exiting
the laboratory; (4) all procedures should be performed carefully to minimize
the creation of aerosols and; (5) mechanical pipetting devices should be
used—mouth pipetting is prohibited.
9
Background
Cloning
Cloning is the production of multiple exact copies of a piece of DNA,
usually a gene, using molecular biology techniques. Cloning is frequently
the first step used in a research project, producing enough DNA for further
study. Once a gene or part of a gene has been amplified using PCR, the
next step is to insert the DNA into a plasmid or cloning vector so that the
DNA fragment can be propagated.
Plasmids as Cloning Vectors
Many cloning vectors are derived from bacterial plasmids. Plasmids are
circular extrachromosomal DNA molecules, usually around 2,000–100,000
base pairs (bp) long, although most plasmids used in cloning are
2,000–10,000 bp. Bacteria may naturally contain many copies of a single
plasmid, or single copies of others. Plasmids are able to replicate
independently of the host DNA and most plasmids carry at least one gene.
Frequently these genes code for a factor or function that helps the bacteria
survive. For example, resistance to the antibiotic ampicillin is conveyed by
a plasmid carrying an ampicillin-resistance gene. Plasmids are capable of
being transferred from one bacterium to another. These characteristics
have resulted both in wonderful new uses for plasmids (such as their use in
cloning, making many of the techniques of molecular biology possible) and
in the emergence of dangerous pathogenic organisms (namely bacteria
resistant to multiple antibiotics).
Plasmids thus already have many of the characteristics needed for use as
cloning vectors, and other useful features have been added through genetic
engineering. A wide variety of vectors are available commercially for various
applications. A plasmid designed to clone a gene is different from a plasmid
designed to express a cDNA (complementary DNA) in a mammalian cell
line, which is different again from one designed to add a tag to a protein for
easy purification. The primary characteristics of any good vector include:
10
• Self-replication — Plasmids have an origin of replication so they can
reproduce independently within the host cell; since the origin of
replication engineered into most cloning vectors is bacterial, the plasmid
can be replicated by enzymes already present in the host bacteria
• Size — Most bacterial vectors are small, between 2,000–10,000 bp
long (2–10 kilobases or kb), making them easy to manipulate
• Copy number — Each plasmid is found at specific levels in its host
bacterial strain. A high copy number plasmid might have hundreds of
copies in each bacterium, while a low copy number plasmid might
have only one or two copies per cell. Cloning vectors derived from
specific plasmids have the same copy number range as the original
plasmid. Most commonly used cloning vectors are high copy number
• Multiple cloning site (MCS) — Vectors have been engineered to contain
an MCS, a series of restriction sites, to simplify insertion of foreign
DNA into the plasmid. An MCS may have 20 or more different enzyme
sites, each site unique both in the MCS and in the plasmid. This
means that for each restriction site included in the MCS, the
corresponding restriction enzyme will cut the plasmid only at its single
site in the MCS
• Selectable markers — Plasmids carry one or more resistance genes
for antibiotics, so if the transformation is successful (that is, if the
plasmid enters and replicates in the host cell), the host cell will grow in
the presence of the antibiotic. Commonly used selectable markers are
genes for resistance to ampicillin (
amp
r
), tetracycline (
tet
r
), kanamycin
(
kan
r
), streptomycin (
sm
r
), and chloramphenicol (
cm
r
)
11
• Screening — When bacteria are being transformed with a ligation reaction,
not all of the religated vectors will necessarily contain the DNA fragment
of interest. To produce visible indicators that cells contain an insert,
vectors frequently contain reporter genes, which distinguish them from
cells that do not have inserts. Two common reporter genes are
beta-galactosidase (b-gal) and green fluorescent protein (GFP)
Some newer plasmid vectors use positive selection, in which the
inserted DNA interrupts a gene that would otherwise be lethal to the
bacteria. If foreign DNA is not successfully inserted into the MCS, the
lethal gene is expressed and transformed cells die. If the foreign DNA
is successfully inserted, the lethal gene is not expressed and the
transformed bacteria survive and divide. Positive selection eliminates
the need for reporter genes, as only cells transformed with vector
containing an insert will survive
• Control mechanism — Most vectors have some control mechanism for
transcription of the antibiotic resistance or other engineered gene. One
of the best-known control mechanisms is the lac operon (an operon is
a group of genes). When lactose (a sugar) is absent in the cell, the lac
repressor protein binds to the lac operon, preventing transcription of the
gene. When lactose is present in the cell, it binds to the lac repressor
protein, causing the repressor protein to detach from the operon. With
the repressor protein no longer bound to the operon, RNA polymerase
can bind and the genes can be transcribed. In this system, lactose acts
as an inducer. (A closely related compound, (IPTG), is often used in the
laboratory as an artificial inducer.) Genes from the lac operon have
been engineered into many cloning vectors
• Size of insert — Plasmid vectors have limitations on the size of inserts
that they can accept, usually less than the size of the vector. Other
vectors have been developed for use if the target DNA is larger, for
example, lambda phage (inserts up to 25 kb), cosmids (inserts up to
45 kb), bacterial artificial chromosomes (BACs; inserts from 100–300 kb),
yeast artificial chromosomes (YACs; inserts from 100–3,000 kb), and
bacteriophage P1 (inserts up to 125 kb)
12
DNA Ligation
Ligation is the process of joining two pieces of linear DNA into a single
piece through the use of an enzyme called DNA ligase. DNA ligase catalyzes
the formation of a phosphodiester bond between the 3'-hydroxyl on one
piece of DNA and the 5'-phosphate on a second piece of DNA.
The most commonly used DNA ligase is T4 DNA ligase (named because it
originated in a bacteriophage named T4). There are several ways that the
efficiency of DNA ligation can be optimized. First, like any enzyme, there
are conditions that are optimal for ligase activity:
• T4 DNA ligase requires ATP and magnesium ions for activity
• The concentration of vector and insert DNA in solution must be high for
efficient ligation
• The molar ratio of insert to vector DNA should be approximately equal,
although the optimal ratio may not be 1:1
13
Chemical structure of deoxyribose sugar and deoxyribose nucleic acid (DNA).
Ligation is used to join vector DNA and insert DNA. There are two ways in
which DNA can be ligated into a cloning vector, one using DNA with
so-called sticky ends and the other using DNA with blunt ends. Unlike DNA
with blunt ends, DNA with sticky ends has one or more unpaired bases at
its ends that do not have complementary bases on the other strand of the
double helix. When a DNA fragment is generated by
Ta q
DNA polymerase
by a process like PCR, it typically has sticky ends with a single adenosine
(A). When a DNA fragment is generated by cutting a piece of DNA with a
restriction enzyme (an enzyme that cuts both strands of double-stranded
DNA), it may have either sticky ends or blunt ends, depending on the
restriction enzyme.
14
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