All living organisms need energy. Energy is the ability to do work. Plants use light energy, carbon dioxide, and water to make sugar energy thorough photosynthesis releasing oxygen.
Endonucleases adhere to the phosphodiester bond making the backbone of each helical strand (deoxyribose and ribose)
Figure 14.13: Sanger sequencing. Following melting of DNA, PCR replication is performed with a small fraction of fluorescent dideoxynucleotides which terminate the polymerization process. Following replication, the strands, each of which terminates with a fluorophore, are separated and the readout gives the sequence.
Did PCR, electrophoresis, and cloning bacterial culture. They ran an agarose gel that first shows the ladders or markers, then the negative controls followed by the positive controls. In the upper area of the gel are the cloning vectors that co-migrate. Then there are the 3 groups of plant samples. First is guava with no cloning since not enough DNA sample was extracted nor amplified as shown in the absence of PCR product band. In sleeping beauty, only two samples were cloned where as oregano brujo was successfully cloned.
1. Cloning and BioinformaticsAnalysesof Novel Plant GAPDH GenesValeria RiveraDr. Michael RubinRISE Program, Department of Biology,University of Puerto Rico at Cayey
2. Background Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) This gene codes for a protein that catalyzes a step of an important pathway, glycolysis, for energy production in carbohydrate metabolism. The human genome has an orthologous GAPDH gene. Techniques used: Polymerase Chain Reaction Electrophoresis Cloning and bacterial culture Plasmid Purification Bioinformatics analyses
6. Significance Study of important genes involved in energy production in plants. Compare GAPDH genes from various plants to study their similarities and differences.
7. Problem Do GAPDH proteins have conserved amino acids related to the active site involved in catalytic function?
8. Hypothesis GAPDH proteins will have conserved amino acids related to the active site involved in catalytic function. GAPDH proteins will have different amino acids at other positions not related to the active site involved in catalytic function.
9. Specific Aims Design PCR Primers and Amplify GAPDH PCR Products Ligate PCR product into plasmid cloning vector pJET Transform Ligation Into competent E. coli Culture Bacteria Purify plasmid DNA, Digest with Restriction Endonucleases to Identify Cloned PCR Products Midi Prep – Large Scale Plasmid Purification Sequence the GAPDH Gene using Sanger Sequencing Bioinformatics Analyses
10. Sanger Sequencing The DNA template is copied. Terminators stop replication (fluorescent nucleotides). Fragments vary in length. Put samples on a plate in the sequencing machine using thin glass capillaries. Electrical field moves the negatively charged DNA to the positive pole through a gel matrix. A laser excites the labeled base creating a fluorescent color that is presented as a color peak.
11. Sanger Sequencing Each color peak represents a sequenced nucleotide. The sequence is determined by reading the different color patterns detected using a laser.