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2011 3.2

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2011 3.2

  1. 1. Jay Gantz, Laflamme Lab Meeting January 13, 2011
  2. 2. What I told you on January 13th...7 4 5 6 8 3 2 1 PolyA PolyA 2A CK7Ins eGFP-Neo hPGK Ins PuroR mCherry Shouldn’t be long now... 3 more easy stepsExposing a beating mTmG syncytium to CK7-CRE lenti is problematic from a visualization standpoint: replate and transduce single cells Will make an EF1a-CRE lenti as a control for mTmG 2
  3. 3. What I told you on January 13th...7 4 5 6 8 3 2 1 PolyA PolyA 2A CK7Ins eGFP-Neo hPGK Ins PuroR mCherry Shouldn’t be long now... 3 more easy stepsExposing a beating mTmG syncytium to CK7-CRE lenti is problematic from a visualization standpoint: replate and transduce single cells Will make an EF1a-CRE lenti as a control for mTmG 2
  4. 4. EF1a-CRE lenti plasmid is done.Virus production is in process with Kara’s help. 3
  5. 5. Beating mTmG cells were successfully replated. CK7-CRE lenti exposure worked as expected.Cells made full red to green transition in ~10-14 days. Need to order antibodies for staining. 4
  6. 6. Staining Plan for mTmG Cells... help? Dish 1 Dish 2Mouse anti-tdTomato Mouse anti-CRE ~$140 ~$250anti-Mouse IgG RFP anti-Mouse IgG RFP Rabbit anti-GFP Rabbit anti-GFP anti-Rabbit IgG GFP anti-Rabbit IgG GFP Chicken anti-cTnT ~$325 Chicken anti-cTnTanti-Chicken IgY YFP anti-Chicken IgY YFPHoechst Counterstain Hoechst Counterstain 5
  7. 7. But do I actually need to stain for tdTomato and eGFP? eGFP Alive After 5’ Triton + 5’ Hoescht After 5’ 4% PF raw photos 2s exposure 6
  8. 8. But do I actually need to stain for tdTomato and eGFP? tdTomato Alive After 5’ Triton + 5’ Hoescht After 5’ 4% PF 7
  9. 9. Merge + Levels adjustmentAlive After 5’ Triton + 5’ Hoescht After 5’ 4% PF 8
  10. 10. So.... cloning.7 4 5 6 8 3 2 1 PolyA PolyA 2A CK7Ins eGFP-Neo hPGK Ins PuroR mCherry 9
  11. 11. So.... cloning.7 4 5 6 8 3 2 1 PolyA PolyA 2A CK7Ins eGFP-Neo hPGK Ins PuroR mCherry Step 6.... just.... didn’t.... work. 9
  12. 12. So.... cloning.7 4 5 6 8 3 2 1 PolyA PolyA 2A CK7Ins eGFP-Neo hPGK Ins PuroR mCherry Step 6.... just.... didn’t.... work. 9
  13. 13. So.... cloning.7 4 5 6 8 3 2 1 PolyA PolyA 2A CK7Ins eGFP-Neo hPGK Ins PuroR mCherry Step 6.... just.... didn’t.... work. New Plan: cut out step 5 replace with new 5+6 PCR from John Mignone 9
  14. 14. So.... cloning.7 4 8 3 2 1 PolyA CK7Ins eGFP-Neo hPGK Ins Step 6.... just.... didn’t.... work. New Plan: cut out step 5 replace with new 5+6 PCR from John Mignone 9
  15. 15. So.... cloning.7 4 5+6 8 3 2 1 PolyA PolyA 2A CK7Ins eGFP-Neo hPGK Ins PuroR mCherry Step 6.... just.... didn’t.... work. New Plan: cut out step 5 replace with new 5+6 PCR from John Mignone 9
  16. 16. It worked... BUTProblem 1 5+6 EcoRI PolyA 2A PuroR mCherry 10
  17. 17. It worked... BUTProblem 1 5+6 EcoRI PolyA 2A PuroR mCherry Homology Arm Cut 1 Homology Arm 10
  18. 18. It worked... BUTProblem 1 5+6 EcoRI PolyA 2A PuroR mCherry Homology Arm Cut 1 Homology Arm Homology Arm Insert 1 Homology Arm 10
  19. 19. It worked... BUTProblem 1 5+6 EcoRI PolyA 2A PuroR mCherry Homology Arm Cut 1 Homology Arm Homology Arm Insert 1 Homology Arm Cut 2 Homology Arm Insert 1 Homology Arm 10
  20. 20. It worked... BUTProblem 1 5+6 EcoRI PolyA 2A PuroR mCherry Homology Arm Cut 1 Homology Arm Homology Arm Insert 1 Homology Arm Cut 2 Homology Arm Insert 1 Homology Arm Homology Arm Insert 2 Insert 1 Homology Arm etc... 10
  21. 21. It worked... BUTProblem 1 5+6 EcoRI PolyA 2A PuroR mCherry 10
  22. 22. It worked... BUTProblem 1 5+6 EcoRI PolyA 2A PuroR mCherry 4 5 6 EcoRI PolyA 2A Homology Arm CK7 PuroR mCherry 7 4 5 6 PolyA 2A CK7 Ins Homology Arm PuroR mCherry 10
  23. 23. It worked... BUT Problem 1 5+6 EcoRI PolyA 2A PuroR mCherrySolution 4 5 6 EcoRI PolyA 2A Homology Arm CK7 PuroR mCherry 7 4 5 6 PolyA 2A CK7 Ins Homology Arm PuroR mCherry 10
  24. 24. It worked... BUT Problem 1 5+6 EcoRI PolyA 2A PuroR mCherrySolution 4 5 6 HindIII EcoRI PolyA 2A Homology Arm CK7 PuroR mCherry 7 4 5 6 PolyA 2A CK7 Ins Homology Arm PuroR mCherry 10
  25. 25. Problem 2 5+6 PolyA XbaI 2A PuroR mCherry 11
  26. 26. Problem 2 5+6 PolyA XbaI 2A PuroR mCherry 7 4 5 6 PolyA XbaI XbaI 2A CK7 InsHomology Arm PuroR mCherry PolyA XbaI XbaI 2A cGata6 InsHomology Arm PuroR mCherry 11
  27. 27. Dam methylationDam methylase recognizes: 5’-GATC-3’ 5’-GAmTC-3’ 12
  28. 28. Dam methylationDam methylase recognizes: 5’-GATC-3’ 5’-GAmTC-3’Xba1: 5’...TCTAGA...3’ 3’...AGATCT...5’ 12
  29. 29. Dam methylationDam methylase recognizes: 5’-GATC-3’ 5’-GAmTC-3’Xba1: 5’...TCTAGA...3’ 3’...AGATCT...5’ 5’...GATCTAGA...3’ 3’...CTAGATCT...5’ 12
  30. 30. Dam methylationDam methylase recognizes: 5’-GATC-3’ 5’-GAmTC-3’Xba1: 5’...TCTAGA...3’ 3’...AGATCT...5’ 5’...GATCTAGA...3’ 5+6 PolyA XbaI 2A 3’...CTAGATCT...5’ PuroR mCherry 12
  31. 31. Solution: Grow plasmid in dam+ bugsHave verified that I only get the CK7 cut out 13
  32. 32. February 2011Questions?

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