a recombinant plasmid contains

If two DNA molecules have matching ends, they can be joined by the enzyme DNA ligase. A process by which the genetic makeup cells is altered by introducing foreign DNA into a bacterial cell. class on Day 2. Helga George Last Modified Date: May 12, 2023 A plasmid is a circular piece of DNA that is found in many bacteria. What is bacterial transformation? sterilize according to Item 3a. Calculate the amount of The pBI-E4 plasmid was cotransfected with linearized pCEP4 (Invitrogen) into 911tet cells. Viruses evolved to be highly efficient at delivering their own genetic information into host cells. Incubate the plates at 37C for 24-36 hours and look for growth. Fill in, Competent E. coli cells (CC) in microfuge tube (Always keep CC tube on ice), Crushed ice in a Styrofoam cup (fill cup first with ice before taking CC tube. Do not open the plates during this step, Each agar plate contains different media- one of Nutrient Agar only (NA), one of Nutrient Agar + ampicillin (NA/AMP), and one of Nutrient Agar + ampicillin + inducer (NA/AMP/IND). 9. provide bacteria with genetic advantages, such as antibiotic resistance. Plasmid vectors for cloning and expression in bacteria (see pUC18 map above) must have. the time you spin the tube. ligated together to form recombinant molecules. plasmids of pAmp and pKan are mixed together with DNA ligase, ATP, immediately into the plates. Spread the P+ cells on the NA, NA/AMP and NA/AMP/IND plates. Repeat this for the NA/AMP plate by resuspending the P- tube cells with the pipette and same tip. Step 1 for Using a Ti plasmid for agriculture. (Image by D. Lee). calcium chloride to the tube. cell at 0C for 24 hours increases the cell competency. 2. 6. Bacteria can also transfer plasmids to one Answered: recombinant plasmid | bartleby The inactivation of one gene on the plasmid while leaving the other gene unchanged is a key requirement for gene cloning. BCH: Chapter 13 Flashcards | Quizlet teacher's direction. 2. Frontiers | Recombinant protein expression in Escherichia coli An additional advantage of using supercoiled adenoviral vectors is that preparation of a single, laboratory-scale batch of the adenoviral vector DNA will yield sufficient material for hundreds of different recombinants. We also acknowledge previous National Science Foundation support under grant numbers 1246120, 1525057, and 1413739. All but 3 ml of the supernatant was removed. vortex to resuspend. Dr. Chois lecture video on recombinant DNA technology (in one 39-min chunk, until I find time to redo this in multiple short segments): I believe the molecular cloning 3 steps for recombination DNA tech. use a commercially prepared 1% solution of ampicillin, add 1 ml of Gene Cloning Part 1: The Mechanics of Recombinant DNA. of sterile, cool distilled water to the 0.05 g of ampicillin salt water and let the tubes cool. the temperature reaches 55C. Modified 2nd and 3rd LOs to be more specific, replaced plasmid cloning figure with new figure that correctly shows sticky ends (using EcoRI as the exemplar restriction enzyme), and two possible products of ligation with foreign DNA fragments. ___ Materials for Iowa teachers are supplied by The Office of In addition, the cell wall is negatively charged and repels negatively charged DNA molecules. We report herein a strategy that builds on several technological and conceptual advances made in the last few years, including alternative systems for producing viral recombinants (1316). A single clone, called 911-E4, was chosen for viral production based on its tight regulation of E4 protein expression. C. Most electrophoretic gels are run horizontally, whether for . The overwhelming majority of researchers believe the technology is not yet precise or accurate enough to attempt edits in human embryos, Introduction to Ecology; Major patterns in Earths climate, Population Genetics: the Hardy-Weinberg Principle, Earth History and History of Life on Earth, Chemical context for biology: origin of life and chemical evolution, Respiration, chemiosmosis and oxidative phosphorylation, Oxidative pathways: electrons from food to electron carriers, Fermentation, mitochondria and regulation, Why are plants green, and how did chlorophyll take over the world? Recombinant DNA | Biological Principles - gatech.edu After a recombinant plasmid enters a bacterial cell, the cell begins to express the genes on it. The mRNA, lacking introns, is a compact version of a eukaryotic gene that retains all of the protein coding information. Describe appropriate methods for cloning eukaryotic genes so their protein products can be expressed (transcribed into mRNA, which is then translated to make protein). 6. All other plasmids used in this study were prepared by CsCl-banding. (Fig.4).4). Blue-white screen - Wikipedia pKan. The pAdTrack-CMV vector is identical to pAdTrack except for the addition of a CMV promoter and polyadenylation site (as in pShuttle-CMV). Cas9 bound to a short RNA can recognize corresponding DNA sequences that match the CRISPR RNAs, after which Cas9 will cleave or cut the DNA, causing a DNA break. For example, the codon TTG codes for the amino acid tryptophan, whereas the codon AAG codes for the amino acid lysine. cell suspension onto the center of a petri dish labeled "LB Amp/Kan," The formation of a recombinant plasmid is taking place in a test tube. ; Some yeast and fungi also contain plasmid along with prokaryotes. As they reproduce, they replicate the plasmid and pass it on to their offspring, making copies of the DNA it contains. Grab a new pipette tip. Close the tube. pressure for the 15 minute period. The Either way, no colonies will form on the media plates. Agar begins Add all solution should be prepared and put in the refrigerator immediately Two percent of a freeze/thaw lysate of these cells was used to infect 293 cells, and fluorescence microscopy of the infected cells was performed 24 hr later. It contains a polylinker for insertion of exogenous transgenes. The ability to recover reasonable quantities of homogeneous viruses, without plaque purification, represents a major practical advantage. to hundreds of thousands of base pairs. the bottom. For some plasmid DNA molecules, only about 1 in 10,000 cells will be transformed. This homology-dependent repair system loops in the sequence information in the homologous DNA template to repair the broken DNA. Please note the following: This page titled 1.13: Transformation is shared under a CC BY 4.0 license and was authored, remixed, and/or curated by Orange County Biotechnology Education Collaborative (ASCCC Open Educational Resources Initiative) . What are some possible reasons for colored colonies to form on the NA/AMP plate? If using the boiling water bath method (3a), it is best LB broth culture, ideally 3-4 hrs before proceeding to the Competent Remove 50 L of the cells. http://www.ibiology.org/ibioeducation/crispr-a-word-processor-for-editing-the-genome.html. Second, the recombination is performed in Escherichia coli rather than in mammalian cells. As shown in Fig. Recombinant DNA technology can readily clone a functional copy of a defective gene and insert it into a vector with the correct regulatory sequences. There are Molecular cloning of eukaryotic genes is often either unfeasible or undesirable, or both, because they contain numerous, large introns. Microcentrifuge the tube at 10,000 RPM for 2 minutes. Prolonged temperatures above 40C These colonies stay white. PacI-digested pAdEasy-GFP-GAL was transfected into 293 cells and GFP expression was visualized by fluorescence microscopy at the indicated times thereafter. In Day 3 of the student lab, the four fragments from the digests are Investigators who wish to obtain any of the vectors described in this work, along with more detailed protocols for their production and analysis, should contact the authors at the following e-mail address: ude.uhj.hclew.knilhclew@ehct. Use a fresh pipette tip for each reagent. ampicillin solution to each 100 ml of LB Amp agar. Step 3. If not, what differences do you see and what are some explanations for these differences? provided by the ISU Office of Biotechnology. destroyed above 60 C. Spread the bacteria on the plate in a zig-zag fragments of 3755 base pairs (bp) and 784 bp, while the pKan digest Plasmid - Wikipedia Gently hold the spreader flat against the surface of the agar; treat it gently as if you were handling gelatin. Second, when GFP expressing cells were marked before staining with X-Gal, every cell that expressed GFP also was found to express -gal and vice versa. Use a sterile plastic pipette to transfer 0.5 ml (10 drops) of Once recombination is achieved and verified, the adenoviral recombinant DNA can be simply transferred to a recA, endA strain (such as DH10B) for greater yields of DNA if desired. The transformation yielded 100 kanamycin-resistant clones, of which two-thirds contained recombinants based on the sizes of undigested miniprep plasmid DNA (Fig. Try to write small along the bottom edge of the plate. (A) DNA from recombinant pAdEasy-GFP+GAL constructs derived from homologous recombination of pAdTrack-CMV-gal and pAdEasy-1in BJ5183 cells was purified from minipreps. kanamycin resistance genes. Multiple copies of the recombinant plasmid can enable the bacterial cell to express large amounts of a protein. (The marked temperature change causes the cells 8. For the best results, the transformation should take place Cell Procedure (Day 2). should begin at least 2-3 days in advance of the laboratory period. Supplies are for each group of three students. students. cell suspension to the center of a dish labeled "LB." This recombinant plasmid contains (1) a promoter that enables transcription of desired gene, (2) a sequence for the initiation of DNA replication ( ori site), and (3) an antibiotic resistance gene. supplies from a scientific supply company. Put the P- and P+ tubes on ice for 15 minutes. Fig.1.1. Remove 10 L of the plasmid and add it to the P+ tube. After sterilizing the agars mixture, the bottle(s) should be swirled Do Be careful to not touch the other end of the spreader to anything but the cells and agar. Overview: DNA cloning (article) | Khan Academy 1 aluminum foil packet containing 6 sterile paper clips that The gene of interest is first cloned into a shuttle vector, e.g., pAdTrack-CMV. Add 50 L of the cells to P- side of the NA/AMP plate using the clamshell method again. Upon completion of this lab, students will be able to: Genetic engineering or DNA technology has been useful for producing large quantities of a specific protein to treat human diseases. No viruses were generated in 12 days after the transfection of circular (i.e., not cleaved with PacI) pAdEasy-GFP+GAL (labeled control). 4. How can bacteria make proteins from foreign DNA, such as human insulin or a protein from a jellyfish like green fluorescent protein (GFP)? If the bacteria lack a resistance gene, the antibiotic will either kill the cell or prevent it from dividing. "pAmp" (store on ice), 5.5 l of 0.20 g /l pKan in a tube marked (Converting light energy into chemical energy), https://www.dnalc.org/resources/3d/20-mechanism-of-recombination.html, http://www.ibiology.org/ibioeducation/crispr-a-word-processor-for-editing-the-genome.html, http://www.cell.com/cell/abstract/S0092-8674(16)31589-6, Creative Commons Attribution-NonCommercial-ShareAlike 4.0 Unported License, Outline the process of molecular cloning of a gene or segment of DNA. before proceeding to the Ligation part (Day 3) of the student The LibreTexts libraries arePowered by NICE CXone Expertand are supported by the Department of Education Open Textbook Pilot Project, the UC Davis Office of the Provost, the UC Davis Library, the California State University Affordable Learning Solutions Program, and Merlot. Plasmids could be produced directly from E. coli BJ5183 cells, but the yields were relatively low (<0.5 g from 2 ml of culture). The https:// ensures that you are connecting to the Cloning into the 5 end of the lacZ gene also means that the E. coli cell can express a protein encoded by the inserted DNA. After transfections of such plasmids into a mammalian packaging cell line, viral production is conveniently followed with the aid of green fluorescent protein, encoded by a gene incorporated into the viral backbone. Allow the "LB No Amp/Kan," "LB Amp," "LB Kan," and "LB Amp/Kan" The next day the gene cloner will observe a mixture of blue and white colonies on the plates. pattern to obtain individual colonies as the concentration of 2. The low efficiency of large fragment ligations and the scarcity of unique restriction sites have made this approach technically challenging. 1.13: Transformation - Biology LibreTexts You make a recombinant plasmid vector that contains an insert of a gene you are interested in studying (see below on left). -bacteriophages Plasmids Those clones that had inserts were further tested by restriction endonuclease digestions, generally PacI, SpeI, and BamHI. The ligation tube, containing the recombinant plasmids you prepared, does not contain any supercoiled plasmids. The maps indicate the cut sites for each restriction enzyme. The plasmid DNA gets into the nucleus, and inserts into random locations in the host cells chromosomes. the teacher to prepare the supplies for the students. After growth in geneticin (0.4 mg/ml, Life Technologies), a clone stably expressing the tet repressor, 911tet, was chosen for further manipulation. The right arm contains Ad5 nucleotides 3,5345,790, which mediate homologous recombination with pAdEasy vectors. 1 petri dish containing LB agar and both antibiotics are out of order. listed from left to right. Take the P-200 pipette, set to 50 L and put on a tip. Sterilely drain off the calcium chloride, being careful not to In an experiment, a scientist attempts to manufacture a protein by inserting a gene of interest, yak-1, into an E. coli cloning vector. All Rights Reserved. two alternatives that can be used. As described in Materials and Methods, a -gal cDNA was placed in the polylinker of pAdTrack-CMV to generate the shuttle vector pGFP+GAL. The alternative to using recombinant plasmids is to directly copy and amplify a specific DNA segment using PCR. Cohen S.N. from the "CaCl2" tube, into the pipette. tips, etc. Step Four: Selecting Bacteria with Recombinant Plasmids You add cells to the surface of the agar (not the plastic lid). Fig.5,5, significant amounts of virus were present as early as 3 days after transfection, concordant with the appearance of first observable viral foci (Fig. In cells transfected with nonlinearized pAdEasy-GFP+GAL, this expression slowly faded over 1 week. recombinant DNA - Recombinant DNA - Cloning, Transformation, Library, Vector DNA: The steps in cloning are as follows. the ligation "Lig" directly to solution using a micropipettor and Open the tube labeled "CaCl2" and add 20 l of Prepare a streak culture plate of MM294 E. coli. 3. (What bacteria will contain a cloned gene, the blue or the white?). Keep all plates right side up for 5 minutes until the liquid is fully absorbed into the plate. Spread the cells from the P tube on your NA and NA/amp plates. refrigerator for later observations. The results obtained while generating a virus encoding -gal provides a representative example of the yields and practical considerations involved with the approach described herein. Take the P+ tube. The reverse transcriptase will also degrade the mRNA from the cDNA:mRNA hybrid molecule. Step Four: Selecting Bacteria with Recombinant Plasmids. Add the 50 L of cells to the P- half of the plate. Figure 13.1 illustrates transformation. Titers determined by plaque assays (expressed in standard plaque-forming units) were equivalent. 50 ml of distilled water in a labeled 100-ml glass bottle with a cap. Therefore, the gene cloner will select white colonies from their plates and assemble a gene library that hopefully has the gene they are interested in. Evidently, degradation of the exogenous DNA or other factors that limit the efficiency of viral production drastically decrease the number of cells that produce virus. Instead of depending on random mutation, and either natural or artificial selection, we now have the ability to directly manipulate the genes of organisms to create new proteins and new capabilities in our domesticated bacteria, fungi, plants and animals. This page has been archived and is no longer updated. In cells transfected with linearized pAdEasy-GFP+GAL, however, this expression never faded and comet-like foci, visualized with GFP fluorescence but invisible by phase contrast microscopy, began to appear at 45 days after transfection (Fig. Place the required volume of distilled water in one or Genes from different organisms can be expressed in other organisms like bacteria since they are encoded in DNA. The starting "parent" plasmids are pAmp and pKan, each of which 7. The dish should be labeled "LB Kan" on the bottom. DNA containing the gene of interest to be cloned is isolated from particular cells/tissues. The ampR gene confers resistance to the antibiotic ampicillin. Their next step is to introduce these plasmids into E.coli bacteria cells. Adenovirus-producing foci after transfection of 293 cells. The students Take the P-200 pipette, set to 50 L and put on a tip. ; Plasmid are small, extra-chromosomal, circular, super-coiled, double stranded DNA molecule present in mostly all species of . If necessary, the ligation can be stored at -20C after Third, there are no ligation steps involved in generating the adenoviral recombinants, as the process takes advantage of the highly efficient homologous recombination machinery present in bacteria. When a bacterium divides, all of the In this lab, we will use a recombinant plasmid as the cloning vector. Both strands are transcribed and nearly all transcripts are heavily spliced. Close the package. The pAdEasy-1 adenoviral plasmid contains all Ad5 sequences except nucleotides 13,533 (encompassing the E1 genes) and nucleotides 28,13030,820 (encompassing E3). Add the antibiotic and pour down (lid down, agar up). CC-BY-SA by Cas9 Wiki project. The Ti plasmid is isolated and prepared for the insertion of the . they also occur in some eukaryotes. Van der Eb of the University of Leiden for generously providing 911 cells, D. Hanahan of the University of California, San Francisco for providing E. coli BJ5183 cells, and P. Hearing of the State University of New York, Stony Brook for providing adenovirus E4ORF6 antibody. A recombinant adenoviral plasmid is generated with a minimum of enzymatic manipulations, using homologous recombination in bacteria rather than in eukaryotic cells. After transformation, bacteria are selected on antibiotic plates. If DNA ligase was added to the tube of cut DNA, the sugar-phosphate backbone bonds would be sealed and the two cut linear DNA molecules would become one circular recombinant plasmid. Copyright Plant and Soil Sciences eLibrary 2023. as long as solutions can be stored in the refrigerator or freezer. Typically, 0.51.0 g of a shuttle vector plasmid (one-fifth of a miniprep) was linearized with PmeI, purified by phenol/chloroform extraction and ethanol precipitation, and mixed with 0.1 g of supercoiled pAdEasy-1 or pAdEasy-2 in a total volume of 6.0 l. Fig.5,5, the efu was 107/ml. procedure. Schematic outline of the AdEasy system. Recombinant DNA - Cloning, Transformation, Library, Vector DNA Streptomyces contain Escherichia coli-type A+T-rich promoters having novel structural features. store until you are ready to use them. 2004 Heredity 92:483. (if appropriate), and paper clips can be accomplished by wrapping The pShuttle plasmid also contains a kanamycin resistance gene from pZero 2.1 (Invitrogen) and the origin of replication from pBR322 (Life Technologies). It disrupts cell wall formation in bacterial cells which kills the cells. Dissolve 0.75 g of CaCl2 in Twenty-microliter aliquots of the electrocompetent BJ5183 cells were kept at 80C. Discard the tip into the biowaste container. Content of Biological Principles at https://sites.gatech.edu/bioprinciples is licensed under a Creative Commons Attribution-NonCommercial-ShareAlike 4.0 Unported License. Fig.33B, the expected restriction fragments were generated in each case. Most separations are done vertically, while sequencing gels are typically run horizontally. up) until the next day at 37 C. 1. that three viable combinations of recombinant plasmids can be Cas9 is a protein enzyme that binds short RNAs made from the CRISPR gene library. and. This recombinant plasmid contains (1) a promoter that enables transcription of desired gene, (2) a sequence for the initiation of DNA replication (ori site), and (3) an antibiotic resistance gene. To chloride treatment and let it sit on ice overnight. Ideally, the mid-log suspension should be prepared 3-4 hours before Compare and contrast the vectors and procedures used for creating genetically modified bacteria and eukaryotes, Describe genome editing with CRISPR-Cas9 what is required for targeting and how target genes can be modified, Cutting DNA at specific sites most often performed by enzymes called, Ligating DNA fragments with an enzyme called, restriction endonuclease sites (polylinker) that occur just once on the vector and allow insertion of cloned DNA segments, a selectable marker gene, such as antibiotic resistance (. preparing four different kinds of media plates for transformation in achieved by streaking an LB plate with bacteria and incubating at The overwhelming majority of researchers believe the technology is not yet precise or accurate enough to attempt edits in human embryos in vivo. Label each plate on the underside, not the lid, Kanamycin solution. Van der Eb of the University of Leiden. The point of creating GMOs is usually to alter one or more traits in the modified organism, most often so they express a new gene. Bacteria colonies that have recombinant plasmids will be white, bacteria with the original, nonrecombinant plasmid will be blue. treatments). Wipe down tabletops with disinfectants and wash hands. The ampicillin solution should not Recombinant DNA - The Cell - NCBI Bookshelf Do a sterile transfer of one pipette full of LB broth into Fig.1.)1.) Incubate the tube for 3-4 hours at 37C. Kpn I I BamH EcoR I I Kpn I Gene W Strategy 1 uses the restriction enzyme KpnI to cut the vector and restriction enzyme W . Prasher D, Eckenrode V, Ward W, Prendergast F, Cormier M. Fallaux F J, Kranenberg O, Creamer S J, Houweling A, van Ormondt H, Hoeben R C, van der Eb A J. Obert S, OConnor R J, Schmid S, Hearing P. Hermeking H, Lengauer C, Polyak K, He T-C, Zhang L, Thiagalingam S, Kinzler K W, Vogelstein B. Hardy S, Kitamura M, Harris-Stansil T, Dai Y, Phipps M L. Proceedings of the National Academy of Sciences of the United States of America, Standard for expression of transgene under CMV promoter, Used only for expression of large genes under CMV promoter, Expression of transgene(s) under a chosen (non-CMV) promoter, Used only for expression of a large gene(s) under a chosen (non-CMV) promoter. 2. 9:Foreign DNA with gene of interest (A, black) and plasmid (B) cut with same restriction enzyme. Importantly, -gal expression perfectly paralleled GFP expression, as assessed in three ways. The most promising techniques use viruses. ampicillin and kanamycin. Place cells on ice in the refrigerator (approx. Several features of adenovirus biology have made such viruses the vectors of choice for certain of these applications. The teacher can remove the tube from the incubator and See text for details. The bottles should not be more than It was critical to linearize the vectors at the PacI sites, as transfection of circular plasmids yielded no viruses, consistent with previous results (14, 23, 24). The presence of an insert can be verified, and its size determined, by restriction analysis of plasmid DNA isolated using a small-scale procedure such as that . 1987; 30:978-982. . DNA polymerase locates the ori- the origin of replication, and starts to replicate the plasmid using the bacterial cells machinery. contains a single recognition site for each enzyme, so each plasmid hold at room temperature overnight. Once you are finished, discard the spreader into the designated biowaste container. The goal is to . In addition, there are informational codons, such as the start codon (ATG) and the stop codon (TTA), which show where in the DNA sequence the code for the protein begins and ends. The alternative to using recombinant plasmids is to directly copy and amplify a specific DNA segment using PCR. Where the foreign DNA meets the vector, there is a cut site for restriction enzyme ABC1. We thank Y.-N. Chang and B. Nelkin for critical reading of the manuscript, A. J. advance of the lab. The recombinant plasmid is named pKC105. Yields were 200600 g per 100 ml of Terrific Broth culture (Life Technologies) for plasmids larger than 30 kb (pAdEasy derivatives) and 4001,000 g for plasmids smaller than 15 kb (shuttle plasmid derivatives). students. Bacterial transformation & selection (article) | Khan Academy Targeted genes (DNA) can be cut with restriction enzymes and joined with other DNA with the enzyme ligase. Always wash your hands for 20 seconds with soap and water before leaving the lab. HHS Vulnerability Disclosure, Help The most notable feature of plasmids is that they replicate independently of the host's main DNA. What would happen if we grew the transformed bacteria in the presence of a different antibiotic such as kanamycin, instead of ampicillin. Recombinants are selected with kanamycin and screened by restriction endonuclease digestion. The control group on nutrient agar with an antibiotic added. These contaminated supplies may include pipette tips, cell spreaders, and microfuge tubes. 5. As soon as the 45 seconds pass, immediately put the tubes back into the ice cup and keep on ice for a minimum of 1 minute. The - mix 20 l of 10X restriction, 6.0 l of BamHI/HindIII enzymes in a tube labeled "B/H" - construct a recombinant plasmid that contains both ampicillin and The transfected and untransfected cells were used as the positive and negative controls respectively . The dish should be labeled "LB Amp/Kan" Usually, a bacterial cell will only make the protein of interest, after it is induced to do so by adding a chemical which will promote the transcription of the gene. which point the bottle(s) can be held without an insulated glove. One day before Competent Cell procedure (Day 2 of the student lab), bottle(s), and alternate boiling and swirling until there are no In CRISPR-Cas9 gene editing, no non-human DNA need be added. will kill the bacteria. Answered: Question 1 A scientist constructs a | bartleby Add Nutrient Broth (NB) to the P- and P+ tubes. These viruses could be used to achieve gene expression in a variety of cell lines of human, mouse, and hamster origin. Keep agar plates closed at all times after removing them from the incubator. ampicillin solution (Step 4) per 100 ml of solution, swirl to mix, ice) labeled "CaCl2, Starter plate of bacteria (prepared on Day 1 of lab), 1 tube of 2X restriction buffer labeled "2X RB", Microcentrifuge (MicroV) or clinical centrifuge, 1 tube 10X ligation buffer/ATP labeled "10X LB/ATP", 5 sterile transfer pipettes- Pasteur or polypropylene. DO NOT FREEZE. The pAdEasy-2 vector is identical to pAdEasy-1 except that it contains an additional deletion of Ad5 nucleotides 32,81635,462 (encompassing E4). plasmid / plasmids. After the cells are heat-shocked, they will be grown under various testing conditions: By examining the growth of bacteria under these conditions, you can verify that your procedure worked, and you can identify the bacteria transformed with the added plasmid. At the theoretical level, the steps needed for obtaining a recombinant protein are pretty straightforward. In Day 1 of the student lab, the plasmids pAmp and pKan are digested in separate reactions with enzymes BamHI and HindIII. Fig.11 and involves three steps. Twenty microliters of electrocompetent E. coli BJ5183 cells were added, and electroporation was performed in 2.0 mm cuvettes at 2,500 V, 200 ohms, and 25 F in a Bio-Rad Gene Pulser electroporator. them. The last feature is important because the ligation of plasmid and foreign DNA segments favors the plasmid ends re-ligating without a foreign DNA insert, resulting in the original, empty plasmid with no foreign DNA insert.
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