Igem2010/Main/Mouse Infection/October

From 2010.igem.org

(Difference between revisions)
(New page: {{:Team:Heidelberg/Template}} {{:Team:Heidelberg/Pagetop|home}} =October= ==15/10/2010== * organize 600 Million HEK293T cells * prepare 2 liter of media ==17/10/2010== * plate 100x 15cm ...)
 
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{{:Team:Heidelberg/Pagetop|home}}
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{{:Team:Heidelberg/Side_Top}}
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=October=
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==15/10/2010==
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* organize 600 Million HEK293T cells
+
-
* prepare 2 liter of media
+
-
==17/10/2010==
+
-
* plate 100x 15cm dishes with each 5.5*10^6 cells
+
-
 
+
-
==18/10/2010==
+
-
* due to contamination 70 new plates are seeded in the afternoon
+
-
==19/10/2010==
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* 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==
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* 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==
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-
* bioluminescence measurements on mice from the first injection round
+
-
 
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-
 
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-
 
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{{:Team:Heidelberg/Pagemiddle}}
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{{:Team:Heidelberg/Side_Bottom}}
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__NOTOC__
 +
=October=
 +
 
 +
===15/10/2010===
 +
* organize 600 Million HEK293T cells
 +
* prepare 2 liter of media
 +
 
 +
===17/10/2010===
 +
* plate 100x 15cm dishes with each 5.5*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/Single_Bottom}}

Latest revision as of 01:05, 27 October 2010

October

15/10/2010

  • organize 600 Million HEK293T cells
  • prepare 2 liter of media

17/10/2010

  • plate 100x 15cm dishes with each 5.5*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

Retrieved from "http://2010.igem.org/Igem2010/Main/Mouse_Infection/October"