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Igem2010/Main/Mouse Infection/October
From 2010.igem.org
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* plate 100x 15cm dishes with each 5.5*10^6 cells | * plate 100x 15cm dishes with each 5.5*10^6 cells | ||
| - | ==18/10/2010== | + | ===18/10/2010=== |
* due to contamination 70 new plates are seeded in the afternoon | * due to contamination 70 new plates are seeded in the afternoon | ||
| - | ==19/10/2010== | + | ===19/10/2010=== |
* triple transfection with PEI using standard protocol for triple transfection: | * triple transfection with PEI using standard protocol for triple transfection: | ||
* set-up of the plate: | * set-up of the plate: | ||
| Line 23: | Line 23: | ||
** 6) tuning construct: perfect binding site for shRNA miRhaat (M1), AAV8, Ad5 | ** 6) tuning construct: perfect binding site for shRNA miRhaat (M1), AAV8, Ad5 | ||
** 7) tuning construct: imperfect binding site randomized from nucleotide 9-12 (M9), AAV8, Ad5 | ** 7) tuning construct: imperfect binding site randomized from nucleotide 9-12 (M9), AAV8, Ad5 | ||
| - | ==21/10/2010== | + | ===21/10/2010=== |
* harvest 70 plates of Hek cells | * harvest 70 plates of Hek cells | ||
* centrifuge: 10 min at 1500 rpm | * centrifuge: 10 min at 1500 rpm | ||
| Line 33: | Line 33: | ||
* soak out the purified virus out of the 40% phase | * soak out the purified virus out of the 40% phase | ||
| - | ==22/10/2010== | + | ===22/10/2010=== |
* double check the titre of capsids via dot plat and the packaged DNA via viral DNA extraction and running on an agarose gel using a virus with a known titre as a control | * double check the titre of capsids via dot plat and the packaged DNA via viral DNA extraction and running on an agarose gel using a virus with a known titre as a control | ||
* mice were injected the following: | * mice were injected the following: | ||
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* 6) Sv40-Luc2-miRhaat perfect binding site double injected with H1-shhaat both in AAV8 capsid | * 6) Sv40-Luc2-miRhaat perfect binding site double injected with H1-shhaat both in AAV8 capsid | ||
* 7) Sv40-Luc2-miRhaat imperfect binding site double injected with H1-shhaat both in AAV8 capsid | * 7) Sv40-Luc2-miRhaat imperfect binding site double injected with H1-shhaat both in AAV8 capsid | ||
| - | ==25/10/2010== | + | ===25/10/2010=== |
*plate 40 15 cm dishes with 5.5*10^6 cells per dish | *plate 40 15 cm dishes with 5.5*10^6 cells per dish | ||
| - | ==26/10/2010== | + | ===26/10/2010=== |
*transfect all 40 plates using PEI transfection buffer and the following set-up | *transfect all 40 plates using PEI transfection buffer and the following set-up | ||
| - | ==27/10/2010== | + | ===27/10/2010=== |
* bioluminescence measurements on mice from the first injection round | * bioluminescence measurements on mice from the first injection round | ||
Revision as of 22:58, 26 October 2010
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