Investigation of genetic modification in maize and soymilk

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  • Main Entry: re·com·bi·na·tion
    Pronunciation: \ˌrē-ˌkäm-bə-ˈnā-shən\
    Function: noun
    : the formation by the processes of crossing-over and independent assortment of new combinations of genes in progeny that did not occur in the parents
  • InstaGene™ matrix: What Function Does It Perform?
    InstaGene matrix consists of a suspension of negatively charged, microscopic beads
    that bind divalent cations such as magnesium (Mg2+). It is important to remove divalent
    cations from the extracted DNA samples because the cations assist enzymes that degrade
    the DNA template. When cheek cells are lysed and boiled in the presence of InstaGene
    matrix, the divalent cations released from the cells bind to the beads, and the heat inactivates
    the DNA-degrading enzymes. The beads are pelleted by centrifugation, and the supernatant,
    which contains clean, intact genomic DNA, can be used as template in PCR reactions.
    The beads in the InstaGene matrix quickly settle out of the suspension. It is therefore
    extremely important that the vial of matrix be thoroughly mixed before pipetting aliquots for
    each student workstation, so that the aliquots contain equivalent amounts of beads.
    If the DNA samples are not going to be amplified within 24 hours, they can be stored in
    the refrigerator in the InstaGene matrix for up to 1 week. For longer storage, place samples
    in the freezer to prevent DNA degradation. Before the samples are used in PCR, the beads
    should be repelleted by centrifugation just prior to making up the PCR reactions.
  • Master Mix: What Is It?
    The master mix contains a mixture of nucleotides, or dNTPs (dATP, dTTP, dCTP, and
    dGTP), buffer, and Taq DNA polymerase. Complete master mix is prepared by adding
    primers to the master mix just prior to the laboratory period. When 20 μl of the DNA template
    is added to 20 μl of complete master mix, all of the necessary components for a 40 μl PCR
    reaction are present.
    Note: Once the master mix and primers are mixed, the complete mix should be kept on ice
    and used within 30 minutes to 1 hr. These reagents are extremely sensitive.
    Why Are the Primers Red and Green?
    The primer mixes contain PCR-compatible dyes that allow students to easily visualize
    and distinguish the different master mixes. The dyes also migrate in the gel giving a visual
    demonstration of electrophoresis.
  • Investigation of genetic modification in maize and soymilk

    1. 1. Frank Soto 5/6/2011
    2. 2.  Background  Genes to identify: 35S promoter, NOS terminator, and PSII  Methodology a) DNA Extraction b) DNA Amplification c) Electrophoresis of DNA  Analysis of Results  Conclusion
    3. 3.  GMO’s are organisms that have been modified through genetic engineering.  In the United States, under guidelines issued by USDA's Animal and Plant Health Inspection Service, genetic engineering is defined as the genetic modification of organisms by recombinant DNA techniques (USDA, 2007)  According to the USDA, crops have been genetically modified ever since 1996.  These crops have all kind of modifications, such as: herbicide tolerance, pesticide tolerance, and bacterial resistance.
    4. 4. Genes Structure of a gene
    5. 5.  Steps for genetic modification of a crop: a) First, find the ideal protein to express. b) Isolation of the gene of interest. c) Insertion of the ideal promoter and terminator d) Back cross to highest-yielding plants
    6. 6.  35S promoter from the cauliflower mosaic virus (CaMV 35S)  Nopaline synthase (NOS) terminator from Agrobacterium Tumefaciens  Photosystem II (PSII)
    7. 7.  Our hypothesis is that the genes 35S and NOS will appear in the gel. The 35S gene will be found at 203bp and NOS at 225bp. We believe the corn will be identified as GMO while the soymilk won’t. In addition, both the corn chips and the papaya will be indentified as GMO.
    8. 8.  DNA Extraction a) Weighing and Grinding of samples b) Pipette 50µL of Slurry into tube containing Instagene matrix. c) Flick tube and place in a 95 degrees celsius water bath for 5 min. d) Place tubes in microcentrifuge for 5 min. Non-GMO Test food Test food 50µL of sample slurry 500µL of Instagene matrix 50µL of sample slurry 500µL of Instagene matrix 50µL of sample slurry 500µL of Instagene matrix
    9. 9.  PCR a) 20µL of indicated master mix is dispensed to each of the samples. b) 20µL of DNA samples are dispensed to their corresponding tubers. c) Once the samples have been dispensed, the tubes are put in the thermal cycler. Non-GMO PMM Non-GMO GMM GMO (+) PMM GMO (+) GMM Test PMM Test GMM Test PMM Test GMM PMM= Plant molecular marker GMM= Genetic molecular marker.
    10. 10.  Electrophoresis a) The gel used was a 3% Agarose gel. b) To prepare for electrophoresis, 10µL of Orange Loading dye was deposited into each of the samples. c) 20µL of Molecular weight ruler(mwr) was deposited to wells to wells 1 and 10. d) 20µL of samples were dispensed to wells 2-9. e) Electrophoresis was run for 30min. at 100V. f) After electrophoresis, the gel was stained with ethidium bromide pads for 20min. 1 Molecular weight Ruler 2 Non-GMO PMM 3 Non-GMO GMM 4 GMO (+) PMM 5 GMO (+) GMM 6 Test PMM 7 Test GMM 8 Test PMM 9 Test GMM 10 Molecular Weight Ruler
    11. 11. Frank’s Gel
    12. 12. Lester’s Gel
    13. 13.  One can conclude that the maize was genetically modified, while the soymilk, papaya, and corn chips are inconclusive.  The experiment must be repeated because the results were affected by human errors, such as bad pipetting technique.

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