Biotechnology Explorer Ligation And . - Bio-Rad
PCR FragmentPCR FragmentBiotechnology Explorer Ligation and TransformationModuleInstruction ManualCatalog #166-5015EDUexplorer.bio-rad.comThis kit is shipped on blue ice. Open immediately upon arrival and storereagents 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 translationsfor Biotechnology Explorer kit curricula.For technical support call your local Bio-Rad office or in the U.S. call 1-800-424-6723.
Table of ContentsIntroduction .1Kit Inventory Checklist .6Safety Issues .9Background .10Quick Guide .26Instructor’s Advance Preparation .32Student Ligation Protocol.36Student Transformation Protocol .40Appendix A Inoculating a Bacterial Colony forPlasmid Miniprep .46Appendix B Restriction Digestion of Plasmid DNAwith Bgl II Enzyme .48
IntroductionCloning is the production of multiple exact copies of a piece of DNA, usuallya gene, using molecular biology techniques. Cloning is frequently the firststep of a research project, producing enough DNA for further study.Using the Ligation and Transformation module students can subclone virtuallyany DNA fragment of interest that has been amplified using PCR. Werecommend that the DNA fragment be approximately 200–2,000 base pairs(bp) in length for best results. Below is a typical workflow for cloning andsequencing a gene. The steps that the Ligation and Transformation moduleenable students to perform are in bold.The Ligation and Transformation module is part of Bio-Rad’s Cloning andSequencing Explorer Series. The Cloning and Sequencing Explorer Seriesis a sequence of individual modules that have been designed to work inconcert to give students the real world experience of a molecular biologyresearch workflow. The additional modules of the Cloning and SequencingExplorer Series can be purchased separately. Further information on theseparate modules is available in the Biotechnology Explorer catalog orfrom explorer.bio-rad.com. Amplify gene of interest using PCR1Purify PCR product2Ligation of PCR product into pJet1.2 plasmidTransform ligated plasmid into bacteriaCulture bacteria and grow minipreps3Purify plasmid from minipreps4Analyze plasmid by restriction digestionElectrophorese restriction digest reaction5Sequence plasmid and analyze61 GAPDH PCR module (catalog #166-5010EDU) amplifies a fragment of the GAPDH genefrom 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 forculturing bacteria for transformation using the Ligation and Transformation module.4 Aurum Plasmid Mini Purification module (catalog #732-6400EDU) contains reagents topurify 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 allowsequencing and bioinformatics analysis of plasmids generated using the Ligation andTransformation module.1
Using the Ligation and Transformation module, students will clone a geneof interest. Prior to starting this laboratory activity, students must havealready amplified a gene of interest using polymerase chain reaction(PCR) and subsequently purified the PCR product to remove excessprimers, nucleotides, and DNA polymerase, which would otherwise interferewith subsequent experiments. Students can then use the Ligation andTransformation module to ligate the DNA fragment into the pJet1.2 bluntedvector, which encodes ampr, an ampicillin-resistance gene. Following ligation,students will perform transformation to introduce the plasmid into livingbacterial cells.The pJet1.2 blunted vector enables positive selection of plasmids with thedesired insert due to the disruption of eco47IR, an otherwise lethal gene,that allows growth of successful transformants. Bacteria are then platedand incubated overnight at 37 C on the selective medium containingampicillin and isopropyl b-D-1-thiogalactopyranoside (IPTG), which isadded to increase expression of the ampr gene. Since transformed cellsexpress an ampicillin-resistance gene, they will grow and divide, eachforming a colony on the plate that is the product of a single transformationevent.The bacteria containing the cloned gene can be grown in liquid growthmedium and the plasmid containing the insert can be purified from thebacteria. The pJet1.2 plasmid contains a Bgl II restriction enzyme recognitionsite on either side of the insertion site. Using the Bgl II enzyme studentswill analyze the cloned plasmid by restriction enzyme digestion and analyzetheir digests by agarose gel electrophoresis to confirm the presence of aninsert and determine its size. The resulting fragment can then be comparedto the size of the PCR fragment ligated into the plasmid. Finally, the DNAfragment can be then sequenced to determine the exact order ofnucleotides in the DNA molecule.What Skills Do Students Need to Perform this LaboratoryActivity?This laboratory activity assumes that students and instructors have basicmolecular biology and microbiology skills, such as proper pipetingtechniques, pouring and streaking agar plates and performing agarose gel2
electrophoresis. In addition, students must understand the principles ofPCR and be able to perform PCR reactions. Bio-Rad’s BiotechnologyExplorer program has a full range of kits to help teach basic skills in individuallaboratories.What Is the Timeline for Completing the Ligation andTransformation Protocol?Before starting this activity, students must have already amplified a geneof interest using PCR. In addition, the PCR product should be purified toremove components of the amplification reaction that would otherwiseinterfere with the ligation step.The amount of time it takes to complete the ligation and transformationprotocols depends greatly on the level of your students and whetheradditional/optional techniques and analyses are performed in addition to thebasic protocol. Steps using the Ligation and Transformation module arehighlighted in bold. Additionally, there are a few incubation steps that addto the number of days it takes to complete the laboratory activity. A roughguide is provided on pages 4 and 5.3
WhenActivity to CompleteDurationAt least 1 day prior toRun a PCR reaction in thermal3–4 hstarting the Ligationcycler to amplify aand Transformationgene of interestmoduleElectrophorese the PCR1hproducts (optional)Purify PCR products0.5 hAt least 3 days prior toPrepare LB and LB Amp0.5 hthe transformation stepIPTG agar platesAt least 2 days prior toPrepare LB broth0.5 hStreak E. coli on a starter LB5 minthe transformation stepagar plateGrow E. coli starter plate at 37 C16 hAs late as possible theInoculate starter culture5 minday before theIncubate starter culture attransformation step37 C in a shaking8 hwater bath or incubatorDay of ligation stepLigate PCR product1hNote: Bolded steps use reagents from the Ligation and Transformationmodule.4
WhenActivity to CompleteDuration1hImmediately followingTransform E. coli with ligationligation or during themixture and plate bacteria onnext laboratory activityLB Amp IPTG agar platesIncubate transformed bacteria16 hat 37 CNext day after theAnalyze results0.5 htransformation stepGrow bacterial colony in LB Amp16 hbroth for miniprepDay after growingPerform miniprep plasmidbacterial culture forpurification to isolateminiprepplasmid carrying insertNext laboratory activityDigest plasmid DNA with1h1hBgl II restriction enzymeAnalyze digest by agarose gel1helectrophoresisPrepare the DNA insert for0.5 hsequencingNote: Bolded steps use reagents from the Ligation and Transformationmodule.5
Kit Inventory ChecklistThis section lists equipment and reagents necessary to perform the ligationand transformation protocol in your classroom or teaching laboratory. Eachkit contains sufficient materials for 12 student workstations, 12 ligationreactions, and 24 transformations. We recommend that students areteamed up – two to four students per workstation. Please use the checklistbelow to confirm inventory.Kit ComponentsNumber/Kit( )T4 DNA ligase, 10 µl1 Ligation reaction buffer (2x concentration), 100 µl1 Proofreading polymerase, 10 µl1 pJet1.2 blunted vector, 10 µl1 Sterile water, 1 ml1 Bgl II restriction enzyme, 50 µl1 10x Bgl II reaction buffer, 1 ml1 Isopropyl b-D-1-thiogalactopyranoside (IPTG), 1 M, 0.1 ml1 Transformation reagent A, 1.25 ml4 Transformation reagent B, 1.25 ml4 C-growth medium, 30 ml1 Microcentrifuge tubes, clear, 1.5 ml30 Microcentrifuge tubes, multicolor, 2.0 ml120 6
Required AccessoriesNumber/Kit( )PCR product (previously amplified andpurified 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) Ampicillin2 vials E. coli HB101 K-12, lyophilized bacteria1 vial Culture tubes, sterile, 15 ml75 Petri dishes, sterile40 Sterile inoculating loops801 pouchVariable speed microcentrifuge (catalog #166-0602EDU)1Shaking water bath or shaking incubator (37 C)1Water bath (catalog #166-0504EDU), heating block,(catalog #166-0562EDU)or incubator (70 C)Adjustable-volume micropipet10.5–10 µl (catalog #166-0505EDU)1220–200 µl (catalog #166-0507EDU)12100–1,000 µl (catalog #166-0508EDU)Pipet Tips120.5–10 µl (catalog #223-9354EDU)1 box2–200 µl (catalog #223-9347EDU)1 box100–1,000 µl (catalog #223-9350EDU)1 boxIce bath1Parafilm sealing film1Marking pens1*Note: Standard microbiological reagents may be used in place of theMicrobial Culturing module (see Instructor’s Advanced Prep section forrequirements). Any E. coli strain commonly used for transformation (forexample, DH5a, DH10, JM107) may be used in place of E. coli HB101.7
Optional AccessoriesVortex mixer (catalog #166-0610EDU)Vacuum sourceAgarose electrophoresis equipmentGAPDH 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 SeparatelyLigation module reagent refill (catalog #166-5016EDU)Includes T4 DNA ligase, 2x ligation reaction buffer, proofreadingpolymerase, pJet1.2 blunted vector, sterile waterBgl II reagent refill (catalog #166-5018EDU)Includes Bgl II restriction enzyme and 10x Bgl II reaction bufferTransformation module reagent refill (catalog #166-5017EDU)Includes transformation reagent A, transformation reagent B, 1 M IPTG,C-growth medium8
Storage InstructionsThe kit is shipped on blue ice. Open immediately upon arrival and storereagent bags immediately at –20 C.Safety IssuesEating, drinking, smoking, and applying cosmetics are not permitted in thework 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 laboratorypractices, including wearing eye protection, gloves, and a laboratory coatto avoid contact with eyes, skin, and clothing. If the solution comes intocontact with gloves, change the gloves. DMSO passes directly throughlatex gloves, readily penetrates skin, and may result in the absorption oftoxic materials and allergens dissolved in the solvent. After handling, washhands 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, respiratorysystem, and skin. In case of contact with eyes, rinse immediately withplenty of water and seek medical advice. Wear suitable protective clothing.Ampicillin is a member of the penicillin family of antibiotics. Those withallergies to penicillin or any other member of the penicillin family ofantibiotics should avoid contact with ampicillin.The E. coli HB101 K-12 strain is not pathogenic. However, handling ofE. coli HB101 K-12 requires the use of standard microbiological practices.These practices include, but are not limited to, the following: (1) work surfacesare 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 involvingorganisms containing recombinant DNA molecules, and (b) before exitingthe laboratory; (4) all procedures should be performed carefully to minimizethe creation of aerosols and; (5) mechanical pipetting devices should beused—mouth pipetting is prohibited.9
BackgroundCloningCloning is the production of multiple exact copies of a piece of DNA,usually a gene, using molecular biology techniques. Cloning is frequentlythe first step used in a research project, producing enough DNA for furtherstudy. Once a gene or part of a gene has been amplified using PCR, thenext step is to insert the DNA into a plasmid or cloning vector so that theDNA fragment can be propagated.Plasmids as Cloning VectorsMany cloning vectors are derived from bacterial plasmids. Plasmids arecircular extrachromosomal DNA molecules, usually around 2,000–100,000base pairs (bp) long, although most plasmids used in cloning are2,000–10,000 bp. Bacteria may naturally contain many copies of a singleplasmid, or single copies of others. Plasmids are able to replicateindependently of the host DNA and most plasmids carry at least one gene.Frequently these genes code for a factor or function that helps the bacteriasurvive. For example, resistance to the antibiotic ampicillin is conveyed bya plasmid carrying an ampicillin-resistance gene. Plasmids are capable ofbeing transferred from one bacterium to another. These characteristicshave resulted both in wonderful new uses for plasmids (such as their use incloning, making many of the techniques of molecular biology possible) andin the emergence of dangerous pathogenic organisms (namely bacteriaresistant to multiple antibiotics).Plasmids thus already have many of the characteristics needed for use ascloning vectors, and other useful features have been added through geneticengineering. A wide variety of vectors are available commercially for variousapplications. A plasmid designed to clone a gene is different from a plasmiddesigned to express a cDNA (complementary DNA) in a mammalian cellline, which is different again from one designed to add a tag to a protein foreasy purification. The primary characteristics of any good vector include:10
Self-replication — Plasmids have an origin of replication so they canreproduce independently within the host cell; since the origin ofreplication engineered into most cloning vectors is bacterial, the plasmidcan be replicated by enzymes already present in the host bacteria Size — Most bacterial vectors are small, between 2,000–10,000 bplong (2–10 kilobases or kb), making them easy to manipulate Copy number — Each plasmid is found at specific levels in its hostbacterial strain. A high copy number plasmid might have hundreds ofcopies in each bacterium, while a low copy number plasmid mighthave only one or two copies per cell. Cloning vectors derived fromspecific plasmids have the same copy number range as the originalplasmid. Most commonly used cloning vectors are high copy number Multiple cloning site (MCS) — Vectors have been engineered to containan MCS, a series of restriction sites, to simplify insertion of foreignDNA into the plasmid. An MCS may have 20 or more different enzymesites, each site unique both in the MCS and in the plasmid. Thismeans that for each restriction site included in the MCS, thecorresponding restriction enzyme will cut the plasmid only at its singlesite in the MCS Selectable markers — Plasmids carry one or more resistance genesfor antibiotics, so if the transformation is successful (that is, if theplasmid enters and replicates in the host cell), the host cell will grow inthe presence of the antibiotic. Commonly used selectable markers aregenes for resistance to ampicillin (ampr), tetracycline (tetr), kanamycin(kanr), streptomycin (smr), and chloramphenicol (cmr)11
Screening — When bacteria are being transformed with a ligation reaction,not all of the religated vectors will necessarily contain the DNA fragmentof interest. To produce visible indicators that cells contain an insert,vectors frequently contain reporter genes, which distinguish them fromcells that do not have inserts. Two common reporter genes arebeta-galactosidase (b-gal) and green fluorescent protein (GFP)Some newer plasmid vectors use positive selection, in which theinserted DNA interrupts a gene that would otherwise be lethal to thebacteria. If foreign DNA is not successfully inserted into the MCS, thelethal gene is expressed and transformed cells die. If the foreign DNAis successfully inserted, the lethal gene is not expressed and thetransformed bacteria survive and divide. Positive selection eliminatesthe need for reporter genes, as only cells transformed with vectorcontaining an insert will survive Control mechanism — Most vectors have some control mechanism fortranscription of the antibiotic resistance or other engineered gene. Oneof the best-known control mechanisms is the lac operon (an operon isa group of genes). When lactose (a sugar) is absent in the cell, the lacrepressor protein binds to the lac operon, preventing transcription of thegene. When lactose is present in the cell, it binds to the lac repressorprotein, causing the repressor protein to detach from the operon. Withthe repressor protein no longer bound to the operon, RNA polymerasecan bind and the genes can be transcribed. In this system, lactose actsas an inducer. (A closely related compound, (IPTG), is often used in thelaboratory as an artificial inducer.) Genes from the lac operon havebeen engineered into many cloning vectors Size of insert — Plasmid vectors have limitations on the size of insertsthat they can accept, usually less than the size of the vector. Othervectors have been developed for use if the target DNA is larger, forexample, lambda phage (inserts up to 25 kb), cosmids (inserts up to45 kb), bacterial artificial chromosomes (BACs; inserts from 100–300 kb),yeast artificial chromosomes (YACs; inserts from 100–3,000 kb), andbacteriophage P1 (inserts up to 125 kb)12
DNA LigationLigation is the process of joining two pieces of linear DNA into a singlepiece through the use of an enzyme called DNA ligase. DNA ligase catalyzesthe formation of a phosphodiester bond between the 3'-hydroxyl on onepiece of DNA and the 5'-phosphate on a second piece of DNA.The most commonly used DNA ligase is T4 DNA ligase (named because itoriginated in a bacteriophage named T4). There are several ways that theefficiency of DNA ligation can be optimized. First, like any enzyme, thereare conditions that are optimal for ligase activity: T4 DNA ligase requires A
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 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 .
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