1 / 22

Extracting Exon0 Isolating a mec-8 Isoform

Extracting Exon0 Isolating a mec-8 Isoform. Sol Katzman. Zahler lab -- C. elegans “Alt splicing, all the time ”. Where’s the binf? Uncoordinated worms Mixing colorless liquids. from http://elegans.swmed.edu/. unc-52 exons 17,18 optional mediated by mec-8. mec-8 four exons +C43002.

price-walters
Download Presentation

Extracting Exon0 Isolating a mec-8 Isoform

An Image/Link below is provided (as is) to download presentation Download Policy: Content on the Website is provided to you AS IS for your information and personal use and may not be sold / licensed / shared on other websites without getting consent from its author. Content is provided to you AS IS for your information and personal use only. Download presentation by click this link. While downloading, if for some reason you are not able to download a presentation, the publisher may have deleted the file from their server. During download, if you can't get a presentation, the file might be deleted by the publisher.

E N D

Presentation Transcript

  1. Extracting Exon0Isolating a mec-8 Isoform Sol Katzman

  2. Zahler lab -- C. elegans“Alt splicing, all the time” • Where’s the binf? • Uncoordinated worms • Mixing colorless liquids from http://elegans.swmed.edu/

  3. unc-52 exons 17,18 optionalmediated by mec-8

  4. mec-8 four exons +C43002

  5. mec-8 zoom out to see Exon0A 6500bp intron in C. elegans?

  6. mec-8 (Wormbase view)

  7. mRNA 3’ 5’ reverse transcribe cDNA 5’ 3’ gsp1 gsp2 5’ RACE procedures cDNA 5’ 3’ purify tail cDNA CCCCC 5’ 3’ AAP PCR dsDNA gsp2 AAP 3’ 5’

  8. SL1 AAP AAP SL1 SL1 tailed tailed ex0for gsp2 gsp2 ex01rev trans-spliced isoforms and primers.known and unknownshould be at least 85bp difference 5’ exon4 exon3 exon2 exon1 3’ 5’ SL1? exon0 3’ exon4 exon3 exon2 exon1

  9. 5’ RACE gel using AAP and SL1 forward primers

  10. TOPO-TA cloning

  11. TOPO clone vector

  12. plasmid digestion after cloning

  13. First clones: exon1 and 2, plus SL1

  14. SL1 AAP AAP SL1 SL1 tailed tailed ex0for gsp2 gsp2 ex01rev trans-spliced isoforms and primers.try exon0forward (and other RNA) 5’ exon4 exon3 exon2 exon1 3’ 5’ SL1? exon0 3’ exon4 exon3 exon2 exon1

  15. Exon0For primer on First Strand cDNA3 RNA samples: embryo, L4, mixed-stageFound something!

  16. mRNA 3’ 5’ reverse transcribe cDNA 5’ 3’ gsp1 gsp2 5’ RACE procedures cDNA 5’ 3’ purify tail cDNA CCCCC 5’ 3’ AAP PCR dsDNA gsp2 AAP 3’ 5’

  17. Exon0For primer: fading through 5’RACE procedure steps. (embryo RNA only)

  18. SL1 AAP AAP SL1 SL1 tailed tailed ex0for gsp2 gsp2 ex01rev trans-spliced isoforms and primers.try exon01reverse primer 5’ exon4 exon3 exon2 exon1 3’ 5’ SL1? exon0 3’ exon4 exon3 exon2 exon1

  19. SL1 forward - Exon01Rev primerson purified cDNA. Double band at 200bp

  20. Clone and PCR sequences – BLAT.2 versions of exon0. SL1 leaders.Where’s the ORF?

  21. To Do • find promoter and bash it • investigate conservation regions in intron • use the third worm -- C. remanei • consider uORFs

  22. Acknowledgements • Al Zahler • Sam Gu • Jen Kabat • Val, Brian, Mary Ann, Poojitha, Joy, Sergio • U.C. Regents

More Related