PCR Fragment PCR Fragment Biotechnology Explorer™ Ligation and Transformation Module Instruction Manual Catalog #166-5015EDU explorer.bio-rad.com 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.
Table of Contents Introduction ..................................................................................1 Kit Inventory Checklist ................................................................6 Safety Issues ................................................................................9 Background ................................................................................10 Quick Guide ................................................................................
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.
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.
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.
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 1h 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 Streak E. coli on a starter LB 5 min the transformation step agar plate Grow E.
When Activity to Complete Duration 1h Immediately following Transform E. coli with ligation 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.
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.
Required Accessories Number/Kit (✔) PCR product (previously amplified and purified by students) 1 per team ❒ 1 ❒ 12 ❒ ❒ ❒ ❒ ❒ ❒ ❒ ❒ ❒ ❒ Microbial Culturing module (catalog #166-5020EDU)* containing the following: • LB broth capsules (each for making 50 ml of LB broth) • LB nutrient agar powder (to make 500 ml) • Ampicillin 2 vials • E.
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, 5
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.
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.
• 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.
• 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.
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.
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.
DNA ligation with sticky ends — To prepare a cloning vector for ligation with insert DNA, it is cut with a restriction enzyme within the MCS, opening it to receive the inserted DNA. If the insert has sticky ends, that is, overhangs on the end of the DNA strands, then the vector should be cut with the same enzyme, producing sticky ends that will be complementary to the ends of the insert DNA.
One advantage to sticky-end ligation is that it makes directional cloning possible. If it is desirable to have the insert in one orientation only (for instance, in the A Æ B direction in the vector, but not in the B Æ A direction), then the insert and vector can both be digested with two different restriction enzymes so that their ends are asymmetric. This is important if the DNA insert is a cDNA encoding a protein to be expressed in the transformed cell.
DNA ligation with blunt ends — Blunt-end ligation, in which both the inserted DNA and the vector have blunt ends, has an advantage compared to sticky-end ligation in that all DNA ends are compatible with all other ends. In other words, it is not necessary to cut the vector and insert with the same restriction enzymes to get complementary overhangs as for sticky-end ligation. Vectors used for blunt-end ligation have a blunt-ended ligation site in the MCS.
• Its MCS has restriction enzyme sites that can be used for later manipulation of the DNA • It is a high copy number plasmid • It contains the b-lactamase gene, ampr, which confers resistance to ampicillin • It contains the eco47IR gene, which allows positive selection of transformants. This gene codes for the Eco47I restriction enzyme, and when the enzyme is expressed, it is toxic to E. coli.
Products of Ligation Ligation is a very inefficient process; from millions of vectors and inserts, only 1–100 are expected to ligate together as desired and lead to growth of colonies from the desired transformants. There are several possible products from a ligation with a vector such as the pJet1.2 blunted vector: • Self-ligation of the vector — A self-ligated vector, without any DNA inserted in the MCS, should have an intact lethal gene.
No interruption of lethal gene so transformed bacteria will die Self-ligation of vector PCR Fragment PCR Fragment Can be minimized by controlling molar ratio of inserts Multiple inserts PCR Fragment PCR Fragment Product cannot replicate Self-ligation of inserts PCR Fragment PCR Fragment Desired product – insert can be in either orientation Ligation of vector and insert Possible ligation products.
• Heat shock is the most easily accomplished transformation method, as it does not require any equipment other than a water bath. Plasmid DNA and heat-shock competent cells in calcium chloride are mixed together and incubated on ice for several minutes. Although the mechanism is not fully understood, calcium chloride causes DNA to bind to the bacterial cell wall. The cells are then subjected to a brief heat shock resulting in the uptake of DNA into the bacteria.
Chamber for cells Metal electrodes Bio-Rad electroporation cuvette. There are ways to increase the number of competent cells in a bacterial culture. To prepare competent cells for heat shock transformation, the bacteria must be washed to remove the growth medium, then resuspended in an ice cold calcium chloride solution. For electroporation, the cells must be washed repeatedly in chilled buffer, and resuspended in a chilled sterile solution that has very low ionic strength.
of the transformation (either the heat shock or the electrical pulse) and begin to express the genes on the plasmid (such as an antibiotic resistance gene), although this step may be omitted. The cells are plated on a selective medium for growth, usually agar plates containing nutrient medium and the antibiotic for which resistance is carried by the plasmid. For example, if the plasmid contains the ampr gene, providing resistance to ampicillin, the agar plates should also contain ampicillin.
Transfer to selective media Plasmid DNA Transformation Incubate at 37°C overnight Transformed cell Pick colonies Competent E. coli Process of bacterial transformation. Competent E. coli are transformed with plasmid DNA. Only a few bacteria take up the plasmid DNA. Bacteria are then plated on selective media and incubated overnight. Only bacteria that contain the plasmid will grow and form colonies. Bacteria colonies are then picked and grown for use in plasmid minipreps.
Looking back at earlier steps in the experiment, a gene or portion of a gene was ligated into the plasmid vector. From previous work, the size of this insert should be known. By digesting a small portion of the miniprep DNA with a specific restriction enzyme such as Bgl II (see below), the insert should be cut out of the vector. Running the products of the restriction digestion on an agarose gel should give two DNA bands, one the size of the vector and the other the size of the inserted DNA.
Ligation – Quick Guide 1. Label a microcentrifuge tube with your initials, plant name, and "ligation." 2. Briefly spin down the stock tubes of 2x reaction buffer and proof reading polymerase to collect the contents at the bottom of the tube. 3. Set up blunting reaction with the following reagents. Reagent Amount 2x ligation reaction buffer 5.0 µl Purified PCR product 1.0 µl Sterile water 2.5 µl Proofreading polymerase 0.5 µl Total 9 µl 4. Close the cap and mix well.
6. Cool tube on ice for 2 min. Ice 7. Once cool, centrifuge briefly to bring contents to the bottom of the tube and keep tube at room temperature. 8. Spin down stock tubes of pJet1.2 vector and the T4 DNA ligase to collect the contents at the bottom of the tube. 9. Set up a ligation reaction with the following reagents. Reagent Amount Blunting reaction (already in microcentrifuge tube) 9.0 µl T4 DNA ligase 0.5 µl pJet1.2 blunted vector 0.5 µl Total 10 µl 10. Close the cap and mix well.
Transformation – Quick Guide Preparation for Competent Cells 1. Label an LB Amp IPTG agar plate with your initials and place at 37°C. 37°C incubator 2. If not already done, pipet 1.5 ml C-growth medium into a 15 ml culture tube. Label with your initials and warm to 37°C for at least 10 min. Also ensure your starter culture is shaking at 37°C. 37°C shaking water bath 3.
7. Centrifuge at top speed for one minute and immediately put tube on ice. Ice 8. Use a 1000 µl pipet or a vacuum source to remove culture supernatant avoiding the pellet. Keep the tube on ice. 9. Resuspend the bacterial pellet in 300 µl of ice cold transformation buffer by very gently pipetting up and down in the solution above the pellet – do not touch the pellet. TF buffer Ice 10. Incubate the resuspended bacteria on ice for 5 min. 11. Centrifuge the bacteria at top speed for 1 min.
Experimental Procedure for Transformation 15. Label one microcentrifuge tube with your initials and "transformation." 16. Pipet 5 µl of your ligation reaction into your transformation microcentrifuge tube. Keep on ice. 17. Using a fresh tip, very gently pipet the competent cells up and down two times then pipet 50 µl of competent cells into your transformation tube. Very gently pipet up and down two times to mix and return to ice. Ice Ligation Competent cells 18.
19. Retrieve the warm LP Amp IPTG agar plate from the 37°C incubator and pipet the entire volume of the transformation onto the labeled agar plate. Use an inoculation loop to very gently spread the bacteria around the plate — do not spread for more than 10 seconds. 20. Immediately place agar plate, upside down at 37°C and incubate overnight. 37°C incubator 21. The next day, analyze results or wrap plate in Parafilm and store at 4°C until required.
Instructor’s Advance Preparation In the first part of this activity, students will insert (ligate) the purified PCR products into the pJet1.2 blunted vector. They will then transform competent bacteria with the ligation reaction mixture. Note: Students can either proceed directly to transformation activity or store the ligation reaction at 4°C or –20°C until the next laboratory session.
Note: Components needed to carry out PCR reactions are not included in the Ligation and Transformation module. A kit that utilizes size exclusion chromatography, such as Bio-Rad’s PCR Kleen™ Spin Purification module (catalog #732-6300EDU), can be used for purifying the PCR products. Once the purified PCR product is available, students may decide to electrophorese the PCR products to assess the quality and quantity of the sample.
relatively high transformation efficiency (106 transformants per µg DNA) without a requirement for a refrigerated centrifuge, commercial competent cells, or a –70°C freezer to store the competent cells. Note: Bio-Rad offers both chemically competent and electrocompetent cells for purchase should your teaching goals include electroporation or more traditional chemical transformation techniques. The commercially available cells require storage at –70°C and have transformation efficiencies of 109/µg DNA.
b. Prepare starter culture: As late as possible the day before the transformation, inoculate a 2–5 ml LB culture with a starter colony from the E. coli starter plate. Incubate cultures in a shaking water bath or incubator overnight at 37°C and at least 200 rpm. Note: It is important to use a fresh starter culture (<24 hours since inoculation) for the transformation, or transformation efficiency will be reduced.
Student Ligation Protocol Note: Before use, the appropriate reagents must be defrosted, thoroughly mixed, and centrifuged to collect contents at the bottom of the tubes. Refer to Tasks to Perform Prior to the Ligation Laboratory on page 33 for details. Listed below are materials and reagents required at the workstations prior to starting the ligation activity.
Setting Up the Blunting Reaction This reaction removes the 3' nucleotide overhang left by the Taq DNA polymerase that would prevent blunt end ligation. 1. Label a microcentrifuge tube with your initials, the name of your amplified gene, and "ligation." 2. Pulse spin the stock tubes containing the ligation reaction buffer and proofreading polymerase in a microcentrifuge for 10 sec to force contents to bottom of tubes prior to use. Note: When pipetting very small volumes, take special care.
4. Close the cap and mix well. Centrifuge in a microcentrifuge for 10 sec to collect the contents at the bottom of the tube. This step is essential due to the very small volume used in this reaction. 5. Place the tube at 70°C for 5 min. 6. Place tube on ice to cool for 2 min. This recondenses water vapor to maintain reaction volume. 7. Once cool, centrifuge the tube briefly to collect the contents at the bottom of the tube. Place tube at room temperature.
11. Incubate tube at room temperature for 5–10 min. 12. Store the ligation reaction at –20°C. If you are proceeding directly to transformation, pipet 5 µl of the ligation reaction into a microcentrifuge tube labeled with your initials, the name of your gene, and "transformation," and store it on ice until needed for the transformation.
Student Transformation Protocol Listed are materials and reagents required at the workstations prior to beginning the transformation activity. Instructor’s (Common) Workstation Quantity (✔) Water bath, heating block, or incubator (37°C) 1 Microcentrifuge (refrigerated, if available) 1 ❒ ❒ Each student team will require the following items to transform bacteria with one PCR product.
Detailed Protocol for Transformation Preparation of Competent Cells 1. Approximately 20–40 min prior to starting the transformation, prepare competent cells by pipeting 150 µl of fresh starter culture (inoculated one day prior) into the prewarmed C-growth medium and placing in a shaking 37°C water bath or incubator for 20–40 min. Note: If a shaking waterbath is not available, manually shake the culture tubes every 5 min during the 20–40 min growth phase to oxygenate the culture.
6. After bacteria have grown in C-growth medium for 20–40 min at 37°C with shaking, transfer the culture to your competent cells tube by decanting or pipetting it. It is better not to put the actively growing cell culture on ice at this step. 7. Centrifuge the culture in a microcentrifuge at top speed for 1 min. Make sure that the microcentrifuge is balanced and accommodate tubes of classmates to ensure economic use of the microcentrifuge. Immediately put the pelleted culture on ice.
9. Resuspend the bacterial pellet in 300 µl of ice-cold transformation buffer by gently pipetting up and down in the solution above the pellet with a 1,000 µl pipet, and gradually wear away the pellet from the bottom of the tube. Make sure that the bacteria are fully resuspended, with no clumps. Avoid removing the cells from the ice bucket for more than a few seconds. 10. Incubate the resuspended bacteria on ice for 5 min. 11. Centrifuge the bacteria in a microcentrifuge for 1 min.
Note: If you are performing the ligation and transformation steps on the same day, use the microcentrifuge containing 5 µl of the ligation mixture that was prepared and labeled at the end of the ligation step. 16. If not already done, pipet 5 µl of the ligation reaction from the previous stage into the "gene TF" microcentrifuge tube. Store any remaining ligation reaction at 4°C or –20°C. 17.
23. Immediately place LB Amp IPTG agar plates upside down in the 37°C incubator and incubate them overnight. 24. The next day, analyze the results, or wrap the plates in Parafilm and place them at 4°C until required for inoculation of miniprep cultures (see Appendix A). Analysis of Results of Ligation and Transformation Count the number of bacterial colonies that grew on the LB Amp IPTG agar plates. Note: Occasionally, satellite colonies may grow using this ligation method.
Appendix A Inoculating a Bacterial Colony for Plasmid Miniprep Once the plasmid has been introduced into living bacterial cells and the cells have grown and divided on selective medium, the next step in the experiment is to prepare a miniprep of the plasmid DNA for sequencing or further experiments.
Note: If no colonies grew on your team’s agar plate from the pJet1.2 + gene ligation reaction, use colonies from another team’s successful transformation. Relabel your 15 ml culture tubes accordingly. 6. Perform a miniprep using the Aurum™ Plasmid Mini Purification module (catalog #732-6400EDU).
Appendix B Restriction Digestion of Plasmid DNA with Bgl II Enzyme Instructors Advanced Preparation The Ligation and Transformation module contains Bgl II enzyme and reaction buffer to enable analysis of plasmids derived using the module and subsequently purified using a plasmid purification protocol. Electrophoresis reagents, including sample loading dye and a molecular weight ruler are also required for the analysis. The pJet1.
By digesting a small portion of the miniprep DNA with Bgl II enzyme (see instructions below), the insert should be cut out of the vector. Running the products of the restriction digestion on an agarose gel should give two DNA bands, one the size of the vector (2,974 bp) and the other the size of the inserted DNA (the original PCR product). If there are more than two bands in any digest, it may mean that the insert contains a Bgl II site.
Reagent Volume for 1 Reaction Volume for 5 Reactions 10x Bgl II reaction buffer 2 µl 10 µl Sterile water 7 µl 35 µl Bgl II restriction enzyme 1 µl 5 µl 10 µl 50 µl Total Experimental Procedure for Restriction Digestion Analysis 1. Label a microcentrifuge tube for each plasmid miniprep. 2. Prepare digestion reactions by combining 10 µl of the Bgl II master mix and 10 µl of each plasmid DNA in the appropriately labeled microcentrifuge tubes. 3.
PCR Fragment BgI II PCR Fragment Bgl II Digest BgI II 1 2 3 Restriction enzyme digestion analysis of plasmid DNA. Circular plasmid DNA purified from bacterial minipreps is isolated and digested with Bgl II, a restriction enzyme producing at least two linearlized fragments — vector DNA and PCR fragment (lane 2). These fragments can be visualized using agarose gel electrophoresis. If the PCR fragment contains a Bgl II restriction site, three DNA bands may be observed (lane 3).
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