Source Of The Recombinant DNA Polymerase SK72 Lab Report

Powerful Essays
3.1 Source of the Recombinant DNA Polymerase SK72
The recombinant E. coli BL21 (DE3) containing the pET 32b/ DNA polymerase SK72 gene was provided by the Laboratory of Enzyme and Microbial Technology, Institute of Bioscience, Universiti Putra Malaysia.
3.2 Preparation of the Lysogeny Broth (LB) Agar Plates
LB agar plates were prepared by dissolving 10g tryptone, 5g yeast extract, 5g NaCl and 15g bacterial agar in 950mL of deionized water. The pH of the medium was adjusted to 7.5 using 1M NaOH before bringing the volume up to 1L. After autoclaving at 121oC for 15 minutes, the medium was cooled to approximately 55oC before 100ug/mL ampicillin was added. The medium was then poured into the petri dishes to let them harden before inverting and storing
…show more content…
Prior to use, the column was equilibrated using 20mM sodium phosphate buffer (pH6.5). The column was washed for 10 CV with the same buffer after the protein loading and the bound proteins were eluted with a linear gradient of NaCl (0-0.5M) in the same buffer over 10 CV at a flow rate of 1mL/min. The negative ions (Cl-) in the salt solution were competed with protein in binding to the resin. Fractions of 2mL were collected and the purity was assessed by 10% SDS-PAGE.
3.5 Determination of the Concentration of the DNA Polymerase SK72
The amount of protein was measured using the commercialized Bradford reagent as described by Bradford (1976). A standard curve was plotted in a range 100-1000ug/mL in a Bradford-compatible buffer using bovine serum albumin as substrate (Appendix A). 1mL of the dye solution was added to 20uL of the protein sample, mixed and incubated for 10 minutes at room temperature before measuring the absorbance at 595nm using the spectrophotometer.
3.6 Verification for the Homogeneity and Molecular Weight of the DNA Polymerase
…show more content…
YASARA program was used to arrange the backbone of the DNA polymerase SK72 identically to the known 3D selected template, which was chosen by virtue of having the highest sequence identity with the targeted sequence. After homology modelling, the model refinement and evaluation of the model quality for correctness of the overall fold and stereochemical parameters like bond lengths and angles was done using Verify3D, Errat Plot and Ramachandran Plot (Bowie et al., 1991 and Colovos et al., 1993). The putative model was used to analyze and interpret the structure, the active site and the special feature of the protein (Ma et al.,

You May Also Find These Documents Helpful

  • Better Essays

    DNA Polymerase Lab Report

    • 929 Words
    • 4 Pages

    this lab was to troubleshoot causes related to PCR components and develop an experiment that would test if the Taq amount is suitable for the PCR reaction to run correctly. INTRODUCTION Thermus aquaticus (Taq) DNA Polymerase is a bacterium that lives in thermophilic conditions and has been identified as an enzyme able to withstand the protein-denaturing conditions (high temperature) required during PCR (Noronha & Mullins, 1992). Why might you not be getting any bands on your PCR? If the Taq DNA polymerase…

    • 929 Words
    • 4 Pages
    Better Essays
  • Good Essays

    RECOMBINANT DNA TECHNOLOGY * It is a technology that allows DNA to be produced via artificial means. * It is the joining together of DNA molecules from two different species that are inserted into a host organism to produce new genetic combinations that are of value to science, medicine, agriculture, and industry. * Since the focus of all genetics is the gene, the fundamental goal of laboratory geneticists is to isolate, characterize, and manipulate genes. Although it is relatively…

    • 482 Words
    • 2 Pages
    Good Essays
  • Good Essays

    Recombinant Dna Technology

    • 1324 Words
    • 6 Pages

    BIOTECHNOLOGY RECOMBINANT DNA technology 1. This is a modern biotechnological advance , in which a desired gene fragment can be inserted in to a cloning vector and the resulting DNA (Recombinant DNA) can be amplified in suitable host. 2. A vector can be a plasmid, cosmid,bacterophage,retroviruses, animal and plant viruses or artificial chromosomes like YAC, BAC,or HAC.(Yeast artificial chromosome, bacterial........) 3. The rec. DNA produced can be amplified or cloned in a suitable vector…

    • 1324 Words
    • 6 Pages
    Good Essays
  • Better Essays

    recombinant dna technology

    • 1093 Words
    • 5 Pages

    Recombinant DNA Technology Recombinant DNA technology refers to the ability to isolate specific DNA sequences and alter or manipulate them to produce desired effects. More often, recombinant DNA technology is referred to as biotechnology. Recombinant DNA technology is fascinating in that it has developed into a multi-billion dollar industry, and completely revolutionized agriculture and pharmaceutical industries, all within the past 50 years. According to one account, biotechnology was born…

    • 1093 Words
    • 5 Pages
    Better Essays
  • Good Essays

    What Is Recombinant Dna

    • 366 Words
    • 2 Pages

    WHAT IS RECOMBINANT DNA So what is rDNA? It stands for Recombinant DNA. Before we get to the “r” part we need to understand DNA. DNA is the keeper of all the information needed to recreate an organism. All DNA is made up of a base consisting of sugar, phosphate and one nitrogen base. There are four nitrogen bases, adenine (A), thymine (T), guanine (G), and cytosine (C).The nitrogen bases are found in pairs, with A & T and G & C paired together. The sequence of the nitrogen bases can be…

    • 366 Words
    • 2 Pages
    Good Essays
  • Good Essays

    So now that we know what recombinant DNA is, we are going to look at its purposes. ******** Recombinant DNA is used to insert a gene for production of a protein of interest The production of heat and drought resistant crops can alleviate world hunger around the world. Production of clotting factors can treat bleeding disorders such a Haemophilia which casues bleeding into the joints and can be life threatening. Hepatitis B vaccines are made with the help of yeast cells It is used for the production…

    • 682 Words
    • 3 Pages
    Good Essays
  • Powerful Essays

    scene by comparing with the DNA samples. Polymerase Chain Reaction(PCR) is used to amplify the small amount of Deoxyribonucleic Acid (DNA) for forensic or genetic studies, which require necessary product and placed in the thermal cycle. Gel electrophoresis is being run in order to analyze and compare the DNA samples at the crime scene with the guilty suspects. Gel electrophoresis is used to separate DNA using an electric current applied to the gel matrix, which causes the DNA samples to move towards…

    • 1917 Words
    • 8 Pages
    Powerful Essays
  • Good Essays

    procedure was taken from “From Drosophila cDNA in E. coli plasmid to homologous human proteins” lab manual (4). - Colony Picking: Two E. coli colonies were grown on agar plates and treated with ampicillin. They contained the plasmid with genes for ampicillin resistance and Drosophila cDNA sequence. - Plasmid Isolation: We used the QuickLyse Miniprep Plasmid DNA purification systems to isolate the plasmid DNA. Indeed, the bacterial cells were removed from the liquid broth and were resuspended in the lysis…

    • 368 Words
    • 2 Pages
    Good Essays
  • Satisfactory Essays

    DNA Lab Report

    • 497 Words
    • 2 Pages

    Biology 110 March 2, 2015 DNA Lab BACKGROUND In this laboratory experiment, students were introduced to DNA electrophoresis. DNA electrophoresis is an instrument that many forensic scientists use to get a DNA fingerprint as an evidence for crimes. Not only can it be used for forensic science, people can use this for paternity test, as well as look for evolutionary relationships among organisms. Agarose is used to make the gel that the DNA fragments are going into. Since DNA particles are negatively…

    • 497 Words
    • 2 Pages
    Satisfactory Essays
  • Better Essays

    Dna Extraction Lab Report

    • 2030 Words
    • 9 Pages

    The Amplification DNA Extraction from minced meat samples using the Polymerase Chain Reaction (PCR) and Gel Electrophoresis for Purification of the DNA. Date: 14th/21st of October 2016 Partner(s): Aisling Loughman. Aim: The aim of the experiment is learn the technique to extract DNA using minced meat samples (Pork, Beef and mixed), amplify the extracted DNA using the PCR Technique and further visualise the extracted DNA by Gel Electrophoresis under UV light. Introduction: “The method…

    • 2030 Words
    • 9 Pages
    Better Essays