1. 6/27/20151 Extracting Exon0 Isolating a mec-8 Isoform Sol Katzman

2. Sol Katzman 6/27/20152 Zahler lab -- C. elegans “Alt splicing, all the time” Where’s the binf? Uncoordinated worms Mixing colorless liquids from http://elegans.swmed.edu/

3. Sol Katzman 6/27/20153 unc-52 exons 17,18 optional mediated by mec-8

4. Sol Katzman 6/27/20154 mec-8 four exons +C43002

5. Sol Katzman 6/27/20155 mec-8 zoom out to see Exon0 A 6500bp intron in C. elegans?

6. Sol Katzman 6/27/20156 mec-8 (Wormbase view)

7. Sol Katzman 6/27/20157 5’ RACE procedures 5’ mRNA 3’ gsp1 reverse transcribe 3’ cDNA 5’ purify 3’ cDNA 5’ tail 3’ cDNA 5’ PCR CCCCC AAP gsp2 5’ dsDNA 3’ AAP gsp2

8. Sol Katzman 6/27/20158 trans-spliced isoforms and primers. known and unknown should be at least 85bp difference exon4exon3exon2exon1 exon4exon3exon2exon1 exon0 SL1 SL1? SL1 AAP 3’ 5’ tailed ex0for ex01revgsp2

9. Sol Katzman 6/27/20159 5’ RACE gel using AAP and SL1 forward primers

10. Sol Katzman 6/27/201510 TOPO-TA cloning

11. Sol Katzman 6/27/201511 TOPO clone vector

12. Sol Katzman 6/27/201512 plasmid digestion after cloning

13. Sol Katzman 6/27/201513 First clones: exon1 and 2, plus SL1

14. Sol Katzman 6/27/201514 trans-spliced isoforms and primers. try exon0forward (and other RNA) exon4exon3exon2exon1 exon4exon3exon2exon1 exon0 SL1 SL1? SL1 AAP 3’ 5’ tailed ex0for ex01revgsp2

15. Sol Katzman 6/27/201515 Exon0For primer on First Strand cDNA 3 RNA samples: embryo, L4, mixed-stage Found something!

16. Sol Katzman 6/27/201516 5’ RACE procedures 5’ mRNA 3’ gsp1 reverse transcribe 3’ cDNA 5’ purify 3’ cDNA 5’ tail 3’ cDNA 5’ PCR CCCCC AAP gsp2 5’ dsDNA 3’ AAP gsp2

17. Sol Katzman 6/27/201517 Exon0For primer: fading through 5’RACE procedure steps. (embryo RNA only)

18. Sol Katzman 6/27/201518 trans-spliced isoforms and primers. try exon01reverse primer exon4exon3exon2exon1 exon4exon3exon2exon1 exon0 SL1 SL1? SL1 AAP 3’ 5’ tailed ex0for ex01revgsp2

19. Sol Katzman 6/27/201519 SL1 forward - Exon01Rev primers on purified cDNA. Double band at 200bp

20. Sol Katzman 6/27/201520 Clone and PCR sequences – BLAT. 2 versions of exon0. SL1 leaders. Where’s the ORF?

21. Sol Katzman 6/27/201521 To Do find promoter and bash it investigate conservation regions in intron use the third worm -- C. remanei consider uORFs

22. Sol Katzman 6/27/201522 Acknowledgements Al Zahler Sam Gu Jen Kabat  Val, Brian, Mary Ann, Poojitha, Joy, Sergio U.C. Regents