Team:Heidelberg/Notebook/Mouse Infection
From 2010.igem.org
(Difference between revisions)
(New page: {{:Team:Heidelberg/Template}} {{:Team:Heidelberg/Pagetop|home}} =Mouse Infection= This page is still under construction. {{:Team:Heidelberg/Pagemiddle}} {{:Team:Heidelberg/Bottom}}) |
(→Mouse Infection) |
||
Line 3: | Line 3: | ||
=Mouse Infection= | =Mouse Infection= | ||
- | + | =October= | |
+ | |||
+ | ==15/10/2010== | ||
+ | * organize 600 Million HEK293T cells | ||
+ | * prepare 2 liter of media | ||
+ | ==17/10/2010== | ||
+ | * plate 100 15cm dishes with each 6*10^6 cells | ||
+ | ==18/10/2010== | ||
+ | * due to contamination 70 new plates are seeded in the afternoon | ||
+ | ==19/10/2010== | ||
+ | * triple transfection with PEI using standard protocol for triple transfection: | ||
+ | * set-up of the plate: | ||
+ | ** 1) off target: construct M23 with perfect binding site for miR122, AAV8 serotype (AAV8), Adenohelper virus 5 (Ad5) | ||
+ | ** 2) control: pBSU6Sv40Luc2 transgene in ITRs transfected together with shuffeled capsid of the 50 clones which was capable of transducing Huh7 and HepG2 cells, Ad 5 | ||
+ | ** 3) control: pBSU6sv40Luc 2 transgene, AAV8, Ad 5 | ||
+ | ** 4) shRNA miR expression vector for miRhaat (pBSU6H1haat), AAV8, Ad 5 | ||
+ | ** 5) the same as 4) | ||
+ | ** 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 | ||
+ | ==21/10/2010== | ||
+ | * harvest 70 plates of Hek cells | ||
+ | * centrifuge: 10 min at 1500 rpm | ||
+ | * wash with 30ml 1xPBS | ||
+ | * centrifuge: 10 min at 1500 rpm | ||
+ | * start freeze and thaw cycles | ||
+ | * pour the gradient | ||
+ | * centrifuge in the ultracentrifuge for 2h at 10°C and 50K | ||
+ | * soak out the purified virus out of the 40% phase | ||
+ | ---- | ||
+ | ==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 | ||
+ | * mice were injected the following: | ||
+ | * 1) SV40-Luc2-miR122 perfect binding site in AAV8 capsid | ||
+ | * 2) SV40-Luc2 in our shuffeled capsid | ||
+ | * 3) SV40-Luc2 in AAV8 capsid | ||
+ | * 4) SV40-Luc2-miRhaat perfect binding site in AAV8 capsid | ||
+ | * 5) SV40-Luc2-miRhaat imperfect binding site (9-12) 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 | ||
+ | ==25/10/2010== | ||
+ | *plate 40 15 cm dishes with 5.5*10^6 cells per dish | ||
+ | ==26/10/2010== | ||
+ | *transfect all 40 plates using PEI transfection buffer and the following set-up | ||
+ | ==27/10/2010== | ||
+ | * bioluminescence measurements on mice from the first injection round | ||
+ | |||
{{:Team:Heidelberg/Pagemiddle}} | {{:Team:Heidelberg/Pagemiddle}} | ||
{{:Team:Heidelberg/Bottom}} | {{:Team:Heidelberg/Bottom}} |
Revision as of 08:50, 24 October 2010
![](https://static.igem.org/mediawiki/2010/9/92/Bg_plasmid.png)
|
|
|||