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

• Purify PCR product

• Ligation of PCR product into pJet1.2 plasmid

• Transform ligated plasmid into bacteria

• Culture bacteria and grow minipreps

• Purify plasmid from minipreps

• Analyze plasmid by restriction digestion

• Electrophorese restriction digest reaction

• Sequence plasmid and analyze

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.

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.

Sequencing and Bioinformatics module (catalog #166-5025EDU) is designed to allow sequencing and bioinformatics analysis of plasmids generated using the Ligation and Transformation module.

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

, 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

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

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.

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.

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.

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 ❒

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.

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

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.

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:

• 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

), tetracycline (

tet

), kanamycin

(

kan

), streptomycin (

), and chloramphenicol (

)

• 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)

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

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.

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