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Part Two

Part Two. Identification of bacteria by DNA sequencing. Sequencing Reaction: One Primer, labeled dNTPs, other PCR reagents. Beckman CEQ 8000 Capillary Head Sequencer. Sequences emailed from facility. FinchTV TM: A free Chromatograph Viewing Program.

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Part Two

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  1. Part Two Identification of bacteria by DNA sequencing

  2. Sequencing Reaction: One Primer, labeled dNTPs, other PCR reagents Beckman CEQ 8000 Capillary Head Sequencer

  3. Sequences emailed from facility. FinchTVTM:A free Chromatograph Viewing Program

  4. Each sample: Analyze overall quality of sequence

  5. Example Chromatogram:

  6. Identify Unknown Bacteria at NCBI: Basic Local Alignment Search Tool

  7. NCBI: A Worldwide Resource                     Origination of some “HITS” to our sequences

  8. RESULTS: Monday Lab 2005

  9. RESULTS: Tuesday Lab 2005

  10. RESULTS:Alignment All Samples Monday Lab Conserved and Variable Regions Generated using ClustalW (free Alignment maker)

  11. RESULTS:Relatedness Alignments are the Basis of Phylogenic Trees 16SrRNA DNA amplified using Primer Set #1 Unrooted tree (generated by Phylip's Drawtree: another online program)

  12. RESULTS:Generating a longer fragment The first primer set amplifies the middle of the 16s DNA, and the second primer set amplifies an end. The sequences overlap so in the future these primers could be used to get a longer sequence. Example Next Slide: Pseudomonas aeruginosa

  13. Primer Set #1 Pseudomonas aeruginosa isolate: 82% identity match with E. coli Region of Alignment PCR sample Full length E. coli 16S rRNA gene ~425 bases of alignable DNA sequence generated

  14. Primer Set #2 Pseudomonas aeruginosa isolate: 83% identity match with E. coli Region of Alignment of PCR Full length E. coli 16S rRNA gene ~280 bases of alignable DNA sequence generated

  15. Class Results on Spreadsheet

  16. Communicating Science: The Real WorldIt is just as important to be able to communicate your results to others as it is to gather and interpret data. • Peer Reviewed Journal Articles • Conferences: • ShortPresentations • Poster Sessions • Informal Networking, Retreats etc. Real Deal

  17. 2) Talks in General: Title: Often good to come up with your own Background: What was the question they were trying to answer? Methods Results: Figures are often the best way to show results Conclusions….. Critiques…. What you would do differently POSITIVE Future directions………….Applications Attention grabber Compare to those we know

  18. 2a) Sample Science Talk: One variant of an Intro to Our Project Methods Some Key Results….not all Attention grabber Are the Bacteria Dangerous? Does this change your view? Discussion + Critique Put in Posters

  19. 2a) Science Talks

  20. 2b) Poster sessions: Adult versions of the Science Fair

  21. Poster session in the parking structure!

  22. Sections for the Poster TITLE(Name; Organization) INTRODUCTION METHODS RESULTSFigures and Tables are good…. Use some we made and Make some of your own Gels, Chromatographs, Alignments etc… Goal: to show a story start to finish in a visual way CONCLUSIONS / Future Research

  23. Making a Poster STANDARD WAY: A Giant Power Point Slide Power Point: under File Menu Choose “Page Set Up” Select Dimensions for Poster Standard Poster Sizes: Width 48 inches Height 36 inches Slides: “landscape” Or Use a Template (steal from a friend) Google “poster template” science; or use link on webpage

  24. Often Easiest to: Put together text inWord or Regular Power Point size slides Cut and paste into the giant slide. Groups for Posters…..

  25. End of semester poster setup and presentations Check talk beforehand Please practice with your group so everyone knows their part

  26. Want to Post Your Sequence? http://www.ncbi.nih.gov/Genbank/index.html

  27. END

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