Bio-rad Ligation and Transformation Module Manuale Utente

Biotechnology Explorer™Ligation and TransformationModuleInstruction ManualCatalog #166-5015EDUexplorer.bio-rad.comFor technical support call your loca

Kit Inventory ChecklistThis section lists equipment and reagents necessary to perform the ligationand transformation protocol in your classroom or tea

Required Accessories Number/Kit (✔)PCR product (previously amplified and 1 per team❒purified by students)Microbial Culturing module (catalog #166-502

Optional AccessoriesVortex mixer (catalog #166-0610EDU) Vacuum sourceAgarose electrophoresis equipmentGAPDHPCR module (catalog #166-5010EDU)PCR Kleen™

Storage InstructionsThe kit is shipped on blue ice. Open immediately upon arrival and storereagent bags immediately at –20°C.Safety IssuesEating, drin

BackgroundCloningCloning is the production of multiple exact copies of a piece of DNA, usually a gene, using molecular biology techniques. Cloning is

• Self-replication — Plasmids have an origin of replication so they canreproduce independently within the host cell; since the origin of replication e

• Screening — When bacteria are being transformed with a ligation reaction,not all of the religated vectors will necessarily contain the DNA fragmento

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

Chemical structure of deoxyribose sugar and deoxyribose nucleic acid (DNA).Ligation is used to join vector DNA and insert DNA. There are two ways inwh

DNA ligation with sticky ends — To prepare a cloning vector for ligationwith insert DNA, it is cut with a restriction enzyme within the MCS, openingit

One advantage to sticky-end ligation is that it makes directionalcloning possible. If it is desirable to have the insert in one orientationonly (for i

DNA ligation with blunt ends — Blunt-end ligation, in which both theinserted DNA and the vector have blunt ends, has an advantage comparedto sticky-en

• 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-lactama

Products of LigationLigation is a very inefficient process; from millions of vectors and inserts,only 1–100 are expected to ligate together as desired

Possible ligation products.TransformationOnce a gene or part of a gene has been amplified using PCR and ligatedinto a plasmid, the next step in clonin

• Heat shock is the most easily accomplished transformation method, asit does not require any equipment other than a water bath. PlasmidDNA and heat-s

Bio-Rad electroporation cuvette.There are ways to increase the number of competent cells in a bacterialculture. To prepare competent cells for heat sh

of the transformation (either the heat shock or the electrical pulse) andbegin to express the genes on the plasmid (such as an antibiotic resistancege

Process of bacterial transformation. Competent E. coliare transformed with plasmid DNA.Only a few bacteria take up the plasmid DNA. Bacteria are then

Looking back at earlier steps in the experiment, a gene or portion of agene was ligated into the plasmid vector. From previous work, the size ofthis i

Table of ContentsIntroduction ...1Kit Inventory Checklist ...

Ligation – Quick Guide1. Label a microcentrifuge tube withyour initials, plant name, and "ligation."2. Briefly spin down the stock tubesof 2

6. Cool tube on ice for 2 min.7. Once cool, centrifuge briefly tobring contents to the bottom ofthe tube and keep tube at roomtemperature.8. Spin down

Transformation – QuickGuidePreparation for Competent Cells1. Label an LB Amp IPTG agarplate with your initials and placeat 37°C.2. If not already done

7. Centrifuge at top speed for oneminute and immediately put tubeon ice.8. Use a 1000 µl pipet or a vacuumsource to remove culture supernatant avoidin

Experimental Procedure forTransformation15. Label one microcentrifuge tubewith your initials and"transformation."16. Pipet 5 µl of your liga

19. Retrieve the warm LP Amp IPTGagar plate from the 37°Cincubator and pipet the entirevolume of the transformation ontothe labeled agar plate. Use an

Instructor’s Advance Preparation In the first part of this activity, students will insert (ligate) the purified PCRproducts into the pJet1.2 blunted v

Note: Components needed to carry out PCR reactions are not included inthe Ligation and Transformation module. A kit that utilizes size exclusionchrom

relatively high transformation efficiency (106transformants per µg DNA)without a requirement for a refrigerated centrifuge, commercial competentcells,

b. Prepare starter culture: As late as possible the day before thetransformation, inoculate a 2–5 ml LB culture with a starter colonyfrom the E. colis

Student Ligation ProtocolNote: Before use, the appropriate reagents must be defrosted, thoroughlymixed, and centrifuged to collect contents at the bo

Setting Up the Blunting ReactionThis reaction removes the 3' nucleotide overhang left by the Ta qDNA polymerase that would prevent blunt end liga

4. Close the cap and mix well. Centrifuge in a microcentrifuge for 10 secto collect the contents at the bottom of the tube. This step is essential due

11. Incubate tube at room temperature for 5–10 min.12. Store the ligation reaction at –20°C. If you are proceeding directly totransformation, pipet 5

Student Transformation ProtocolListed are materials and reagents required at the workstations prior tobeginning the transformation activity. Instructo

Detailed Protocol for TransformationPreparation of Competent Cells1. Approximately 20–40 min prior to starting the transformation, preparecompetent ce

6. After bacteria have grown in C-growth medium for 20–40 min at 37°Cwith shaking, transfer the culture to your competent cells tube bydecanting or pi

9. Resuspend the bacterial pellet in 300 µl of ice-cold transformationbuffer by gently pipetting up and down in the solution above the pelletwith a 1,

44Note: If you are performing the ligation and transformation steps on thesame day, use the microcentrifuge containing 5 µl of the ligation mixtureth

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

IntroductionCloning is the production of multiple exact copies of a piece of DNA, usuallya gene, using molecular biology techniques. Cloning is freque

Appendix AInoculating a Bacterial Colony for PlasmidMiniprepOnce the plasmid has been introduced into living bacterial cells and thecells have grown a

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 transformat

Appendix BRestriction Digestion of Plasmid DNA withBgl II Enzyme Instructors Advanced PreparationThe Ligation and Transformation module contains Bgl

By digesting a small portion of the miniprep DNA with Bgl II enzyme (seeinstructions below), the insert should be cut out of the vector. Running thepr

Reagent Volume for 1 Reaction Volume for 5 Reactions10x Bgl II reaction buffer 2 µl 10 µlSterile water 7 µl 35 µlBgl II restriction enzyme 1 µl 5 µlTo

Restriction enzyme digestion analysis of plasmid DNA. Circular plasmid DNA purified frombacterial minipreps is isolated and digested with Bgl II, a re

Legal NoticesNotice regarding Bio-Rad thermal cyclers and real-time systems: Purchase ofthis instrument conveys a limited, non-transferable immunity f

Using the Ligation and Transformation module, students will clone a geneof interest. Prior to starting this laboratory activity, students must havealr

1665019 Rev ALife ScienceGroup00-0000 0000 Sig 0211Bulletin 0000 Rev A US/EGBio-Rad Laboratories, Inc.Web site www.bio-rad.com USA 800 424

electrophoresis. In addition, students must understand the principles ofPCR and be able to perform PCR reactions. Bio-Rad’s BiotechnologyExplorer prog

When Activity to Complete DurationAt least 1 day prior to Run a PCR reaction in thermal 3–4 h starting the Ligation cycler to amplify a and Transfor

When Activity to Complete DurationImmediately following Transform E. coli with ligation 1 hligation or during the mixture and plate bacteria on next