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    Cloning Lab Final Doc 1

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    Emily Linn and Lucy Kurtz cloned the kanr gene for kanamycin resistance using DNA cloning technology. They followed the Edvotek 301 standard cloning protocol, which involves ligating the kanr gene fragment into a plasmid vector, transforming E. coli with the recombinant DNA, culturing the transformed bacteria, and extracting and analyzing the replicated plasmid DNA. Most steps were successful except the transformation and restriction digest analysis. The DNA was recombined but the digest results were inconclusive, likely due to incorrect reaction conditions. This cloning experiment demonstrates how genes can be artificially replicated using bacterial cells.

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    Cloning Lab Final Doc 1

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    120 views11 pages
    Emily Linn and Lucy Kurtz cloned the kanr gene for kanamycin resistance using DNA cloning technology. They followed the Edvotek 301 standard cloning protocol, which involves ligating the kanr gene fragment into a plasmid vector, transforming E. coli with the recombinant DNA, culturing the transformed bacteria, and extracting and analyzing the replicated plasmid DNA. Most steps were successful except the transformation and restriction digest analysis. The DNA was recombined but the digest results were inconclusive, likely due to incorrect reaction conditions. This cloning experiment demonstrates how genes can be artificially replicated using bacterial cells.

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    Emily Linn and Lucy Kurtz cloned the kanr gene for kanamycin resistance using DNA cloning technology. They followed the Edvotek 301 standard cloning protocol, which involves ligating the kanr gene fragment into a plasmid vector, transforming E. coli with the recombinant DNA, culturing the transformed bacteria, and extracting and analyzing the replicated plasmid DNA. Most steps were successful except the transformation and restriction digest analysis. The DNA was recombined but the digest results were inconclusive, likely due to incorrect reaction conditions. This cloning experiment demonstrates how genes can be artificially replicated using bacterial cells.

    Copyright:

    © All Rights Reserved
    Download as DOCX, PDF, TXT or read online from Scribd
    Download as docx, pdf, or txt
    120 views11 pages

    Cloning Lab Final Doc 1

    Uploaded by

    api-285365116
    Emily Linn and Lucy Kurtz cloned the kanr gene for kanamycin resistance using DNA cloning technology. They followed the Edvotek 301 standard cloning protocol, which involves ligating the kanr gene fragment into a plasmid vector, transforming E. coli with the recombinant DNA, culturing the transformed bacteria, and extracting and analyzing the replicated plasmid DNA. Most steps were successful except the transformation and restriction digest analysis. The DNA was recombined but the digest results were inconclusive, likely due to incorrect reaction conditions. This cloning experiment demonstrates how genes can be artificially replicated using bacterial cells.

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    CONSTRUCTION AND CLONING OF A RECOMBINANT DNA

    Emily Linn and Lucy Kurtz


    ABSTRACT
    In this lab, we used DNA cloning technology to replicate the kanr gene for kanamycin
    resistance. This technology is a relatively new and extremely relevant process in biomedical
    research today. Paul Berg did the first successful application of the technology in 1971. In our
    study, we used the steps outlined in the Edvotek 301 standard cloning and transformation
    protocol. We found success in each step with the exception of Module II and Module V. Our
    DNA was successfully recombined, however, the digest results were inconclusive according to
    the possible orientations.
    INTRODUCTION
    Genetic cloning technology is a relatively new and extremely relevant process in
    biomedical research today. The first successful application of recombinant DNA technology was
    in 1971, when Paul Berg successfully spliced bacterial lamba DNA into the DNA of simian virus
    SV40 (BioTech, 2016). The process of digestion and ligation employed by Berg is the same
    technology used in this cloning experiment. Plasmids, usually made up of 2,000 to 10,000 base
    pairs, are digested by a restriction enzyme, and a fragment is typically inserted between the
    sticky ends where the cut is made (Slish, 1999). Between insertion and replication, the samples
    of digested DNA must be run through a gel to determine success. Then, DNA ligases are added
    to the sample that catalyze the formation of a phosphodiester bond between the 3' hydroxyl and
    5' phosphate of the adjacent DNA residues (BioLabs, 2016). The plasmid is then left to replicate
    within the bacteria as it goes through binary fission. Recombinant DNA works when the host cell

    expresses protein from the recombinant genes (Kuure-Kinsey et. all, 2000). For this reason,
    bacteria must be tested to ensure that only those that contain the vector survive. They are placed
    on plates where only antibiotic-resistant bacteria grow, and the only bacteria with resistance are
    those that took up the inserted fragment as well. The cloned genes can then be collected in a lab.
    In Our Study, we used E. coli vectors. These plasmids are commonly used because they can be
    simplified by reducing their length (Griffiths et. all, 1999). The fragment that we are adding to be
    replicated is the kanr gene for kanamycin resistance.

    METHODS

    We used the Edvotek 301 standard cloning and transformation protocol. The basic steps
    of the protocol are outlined below:
    o Module I
    I-A: Ligation of the Plasmid Vector to the kan^R Gene Fragment
    Stock ligation reaction mixture is added to a ligation control tube (C1) and

    a T4 DNA ligase tube.


    Sample are incubated and half of DNA ligase is added to ligation reaction

    tube (R1).
    Loading gel solution is added to C1 and R1 to be analyzed on a gel.
    T4 DNA ligase tube moves to Module II for transformation.
    I-B: Agarose Gel Electrophoresis
    Agarose gel is made.
    I-C: Staining with InstaStain Ethidium Bromide
    Gel is stained with FlashBlue stain.
    Gel is analyzed once stain is finished.
    o Module II: Transformation of the Recombinant DNA into E.coli
    E.coli colonies are added to CaCl2 and suspended into the solution.
    Solution is then transferred to a ligation reaction tube (R2) and a control
    reaction tube (C2).

    Diluted Ligation Rxn DNA is added to R2 and control DNA is added to

    C2.
    R2 and C2 are spread on two plates for colonies to form.
    o Module III: Culturing of kan^R Transformants
    One single isolated colony is added to a liquid medium containing
    Kanamycin antibiotic.
    Solution is then incubated overnight and a pellet is formed.
    o Module IV: Extraction of Recombinant Plasmid DNA
    Buffer is added to the pellet and pellet is resuspended into the solution.
    A lysis buffer is then added to the solution and then mixed.
    Potassium Acetrate Solution is added to the mixture and then mixed to

    form a white precipitate.


    Supernatant is transferred to a clean tube and 100% isopropanol is added

    and mixed.
    Solution is centrifuged to form pellet and then washed.
    Pellet is resuspended in a 1x Tris-EDTA buffer.
    o Module V
    V-A: Restriction Enzyme Analysis
    Restriction Digest Cocktail is made and added to tubes 3, 4, 5, and 6.
    Ultra-pure water is added to each tube.
    EcoRI is added to tube 4.
    PvuII is added to tube 5.
    ClaI is added to tube 6.
    Tubes are prepared for electrophoresis.
    V-B: Analysis of Restriction Digests by Electrophoresis
    DNA samples are added to the wells and electrophoresis is ran using he
    same steps in Module I-C.
    RESULTS
    o Module I
    R1 and C1 tubes to be analyzed on a gel

    T4 DNA Ligase tube to be sent to transformation

    A stained final gel with ligation results

    o Module II

    o Module III

    A control plate with colonies and ligation reaction plate with colonies

    o Module IV

    Tube containing bacterial colonies and Kanamycin solution

    Tube with resuspended pellet in a 1x Tris-EDTA buffer

    o Module V
    4 tubes
    3 Control
    4 EcoRI
    5 PvuII
    6 ClaI
    Gel with final solution of cloned DNA

    Lane

    Sample

    Result

    DNA Standard
    Marker

    Molecular Weight
    (bp)
    6751, 3652,
    2827,1568, 1118,
    825, 630

    Supercoiled
    Plasmid Vector

    4300

    Standard
    Restriction Digest
    Control
    EcoRI Digest
    PvuII Digest
    PvuII/ClaI

    3
    4
    5
    6

    3600

    2
    1
    2

    4250, 3500
    4000
    3650, 3500

    DISCUSSION
    Each step was successful with the exception of Module II and Module V. We believe we
    either did not have enough E.coli in our tubes and we incorrectly spread the solution on the
    plates. Therefore, there was not any growth on our plates. Our final gel had bands incongruent
    with the restriction digest most likely because of digestion at incorrect temperatures and/or
    inappropriate amounts of time. However, we believe it was closest to a Single Insert 5-3
    orientation. This technology is typically used in labs in a more intensive form. The gene we
    inserted only codes for antibiotic resistance, and is usually used in standard lab procedures as a
    tell for successful recombination of another target DNA sequence. These sequences typically
    code for proteins or enzymes of medical importance, such as insulin. Final products can either be
    transcribed for pharmaceutical companies or further amplified for scientific use.

    LITERATURE CITED

    BioLabs. 2016. DNA Ligation. https://www.neb.com/applications/cloning-and-synthetic-

    biology/dna-ligation. Date accessed (03/09.16).


    BioTech. 2016. Recombinant DNA. https://biotechhistory.org/timeline/recombinant-dna/.

    Date accessed (03/09.16).


    Griffiths, A.J.F., W.M. Gilbert, J.H. Miller, and R.C. Lewontin. 1999. Making
    Recombinant DNA - Modern Genetic Analysis.

    http://www.ncbi.nlm.nih.gov/books/NBK21407/. Date accessed (03/09.16).


    Kuure-Kinsey, M. and B. McCooey. The Basics of Recombinant DNA.
    http://www.rpi.edu/dept/chem-eng/Biotech-Environ/Projects00/rdna/rdna.html. Date

    accessed (03/09.16).
    Slish, D. 1999. Cell Biology Lab.
    http://faculty.plattsburgh.edu/donald.slish/LabSyllabus.html. Date accessed (03/09.16).

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