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Gapdh research august 2009

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Gapdh research august 2009

  1. 1. PCR AMPLIFICATION, CLONING, SEQUENCE DETERMINATION, AND BIOINFORMATICS ANALYSES OF NOVEL PLANT GAPDH GENES FROM CYPERUS ALTERNIFOLIUS , SCHEFFLERA ACTINOPHYLLA AND TROPICAL FLORA ENDEMIC TO PUERTO RICO Lydia E. Cortes and Dr. Michael Rubin Department of Biology and RISE Program University of Puerto Rico at Cayey
  2. 2. GAPDH <ul><li>G lycer a ldehyde-3- p hosphate d e h ydrogenase </li></ul><ul><li>Is a protein coding gene </li></ul><ul><li>Serves to break down glucose for energy and carbon molecules </li></ul><ul><li>Has recently been implicated with transcription activation and initiation of apoptosis, and also shuttling between Endoplasmic Reticulum and Golgi vesicle. </li></ul>
  3. 3. CYPERUS ALTERNIFOLIUS and SCHEFFLERA ACTINOPHYLLA <ul><li>Cyperus alternifolius </li></ul><ul><ul><li>umbrella papyrus or umbrella palm </li></ul></ul><ul><ul><li>is a grass-like plant </li></ul></ul><ul><ul><li>has eight genes sequenced </li></ul></ul><ul><li>Schefflera actinophylla </li></ul><ul><ul><li>Brassaia actinophylla </li></ul></ul><ul><ul><li>is a tree </li></ul></ul><ul><ul><li>five genes sequenced </li></ul></ul>
  4. 4. PROBLEM <ul><li>Are GAPDH genes conserved between Cyperus alternifolius and Schefflera actinophylla and other sequenced GAPDH genes? </li></ul><ul><li>What amino acid regions are similar and different between the two plants? </li></ul>
  5. 5. SIGNIFICANCE <ul><li>By sequencing the gene it will be possible to study its sequence and mutations, information that could be used in future studies. </li></ul><ul><li>By finding the similar and different sequences between these two plants it will be possible to find the evolutionary relation between each plant and other plants where this gene is sequenced. </li></ul>
  6. 6. SPECIFIC AIMS <ul><li>Transform and clone Cyperus alternifolius and Schefflera actinophylla GAPDH genes into E. Coli JM109. </li></ul><ul><li>Purify plasmid DNA containing cloned Cyperus alternifolius and Schefflera actinophylla GAPDH genes. </li></ul><ul><li>Determine the sequence of cloned Cyperus alternifolius and Schefflera actinophylla GAPDH genes. </li></ul><ul><li>Bioinformatics analysis of Cyperus alternifolius and Schefflera actinophylla GAPDH genes. </li></ul>
  7. 7. HYPOTHESIS <ul><li>The sequence of the GAPDH gene should be similar in some sections, but at the same time they must have some differences. </li></ul>
  8. 8. EXPERIMENTAL DESIGN <ul><li>Transform and clone GAPDH genes into E. Coli JM109 </li></ul>1 ul DNA 20 ul of competent cells 42 ° C 90 seconds 2 minutes 950 ul Of SOC media 30 minutes
  9. 9. CONTINUATION OF TRANSFORMATION Amp plates- 900 and 50 ul Overnight (both) LB Broth + Amp solution (100 ul of Amp and 100 ml of LB Broth)
  10. 10. PLASMID PURIFICATION Resuspend in 200ul resuspension solution 250 ul lysis solution 250ul neutralization solution
  11. 11. CONTINUATION PLASMID PURIFICATION Move to tube With column Add 200 ul matrix 2 minutes 500 ul Wash solution 100 ul water 1 minute
  12. 12. SEQUENCE DETERMINATION
  13. 13. CONTINUATION OF SEQUENCE DETERMINATION 10 ul of DNA + 1 ul of primer Pipet 10 ul Seal and mail
  14. 14. BIOINFORMATICS ANALYSIS <ul><li>CAP 3 </li></ul>
  15. 15. CONTINUATION BIOINFORMATICS <ul><li>Predicting mRNA sequence </li></ul><ul><li>Predict protein sequence </li></ul>
  16. 16. PRELIMINARY RESULTS: CLONING GEL No DNA pGAP Arabidopsis Marker Cyperus Marker Schefflera
  17. 17. PRELIMINARY RESULTS OF TRANSFORMATION Type of DNA Number of cells in 50 microliters Number of cells in 900 microliters CYPERUS ALTERNIFOLIUS 17 440 SCHEFFLERA ACTINOPHYLLA 10 560
  18. 18. ACKNOWLEDGEMENTS <ul><li>Dr. Robert Ross </li></ul><ul><li>Dr. Edgar Llera </li></ul><ul><li>Yadira Ortiz </li></ul><ul><li>RISE Students: Mayrim Bernard, Aixa Castro, Sheydanis Díaz, Luis L ó pez, and Pedro Rodríguez </li></ul><ul><li>Melisa Medina </li></ul><ul><li>Paola Montes </li></ul><ul><li>Ana Velazquez </li></ul><ul><li>RISE Program (R25GM59429) </li></ul>
  19. 19. PCR AMPLIFICATION, CLONING, SEQUENCE DETERMINATION, AND BIOINFORMATICS ANALYSES OF NOVEL PLANT GAPDH GENES FROM CYPERUS ALTERNIFOLIUS , SCHEFFLERA ACTINOPHYLLA AND TROPICAL FLORA ENDEMIC TO PUERTO RICO Lydia E. Cortes and Dr. Michael Rubin Department of Biology and RISE Program University of Puerto Rico at Cayey

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