Jay Gantz, Laflamme Lab Meeting        January 13, 2011
What I told you on January 13th...7      4             5                  6 8 3                    2        1             ...
What I told you on January 13th...7      4             5                  6 8 3                    2        1             ...
EF1a-CRE lenti plasmid is done.Virus production is in process with Kara’s help.                       3
Beating mTmG cells were successfully replated.   CK7-CRE lenti exposure worked as expected.Cells made full red to green tr...
Staining Plan for mTmG Cells... help?       Dish 1                        Dish 2Mouse anti-tdTomato             Mouse anti...
But do I actually need to stain for tdTomato and eGFP?                       eGFP   Alive                      After 5’ Tr...
But do I actually need to stain for tdTomato and eGFP?                     tdTomato   Alive                      After 5’ ...
Merge + Levels adjustmentAlive                        After 5’ Triton + 5’ Hoescht          After 5’ 4% PF                ...
So.... cloning.7      4            5                  6 8 3                    2        1                                 ...
So.... cloning.7      4              5                  6 8 3                    2        1                               ...
So.... cloning.7      4              5                  6 8 3                    2        1                               ...
So.... cloning.7      4              5                  6 8 3                    2        1                               ...
So.... cloning.7      4                             8 3             2        1                                          Po...
So.... cloning.7      4                5+6                      8 3              2        1                               ...
It worked... BUTProblem 1                          5+6                                   EcoRI                            ...
It worked... BUTProblem 1                                  5+6                                            EcoRI           ...
It worked... BUTProblem 1                                  5+6                                                EcoRI       ...
It worked... BUTProblem 1                                  5+6                                                EcoRI       ...
It worked... BUTProblem 1                                  5+6                                                  EcoRI     ...
It worked... BUTProblem 1                          5+6                                   EcoRI                            ...
It worked... BUTProblem 1                                  5+6                                                EcoRI       ...
It worked... BUT Problem 1                                   5+6                                                 EcoRI    ...
It worked... BUT Problem 1                                    5+6                                                  EcoRI  ...
Problem 2                          5+6                                          PolyA                                   Xb...
Problem 2                                   5+6                                                                  PolyA    ...
Dam methylationDam methylase recognizes:       5’-GATC-3’          5’-GAmTC-3’                      12
Dam methylationDam methylase recognizes:        5’-GATC-3’          5’-GAmTC-3’Xba1:    5’...TCTAGA...3’    3’...AGATCT......
Dam methylationDam methylase recognizes:        5’-GATC-3’           5’-GAmTC-3’Xba1:    5’...TCTAGA...3’    3’...AGATCT.....
Dam methylationDam methylase recognizes:        5’-GATC-3’           5’-GAmTC-3’Xba1:    5’...TCTAGA...3’    3’...AGATCT.....
Solution:        Grow plasmid in dam+ bugsHave verified that I only get the CK7 cut out                     13
February 2011Questions?
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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?

    ×