Team:Heidelberg/Notebook/Mouse Infection/October

From 2010.igem.org

(Difference between revisions)
(New page: {{:Team:Heidelberg/Single}} {{:Team:Heidelberg/Single_Pagetop|note_virobytes}} {{:Team:Heidelberg/Side_Top}} {| cellpadding="5" cellspacing="0" style="text-align: center; color:#009be1; bo...)
Line 1: Line 1:
{{:Team:Heidelberg/Single}}
{{:Team:Heidelberg/Single}}
-
{{:Team:Heidelberg/Single_Pagetop|note_virobytes}}
+
{{:Team:Heidelberg/Single_Pagetop|note_mouse_infection}}
{{:Team:Heidelberg/Side_Top}}
{{:Team:Heidelberg/Side_Top}}
-
{| cellpadding="5" cellspacing="0" style="text-align: center; color:#009be1; border: 3px solid #009be1;"
+
{| cellpadding="5" cellspacing="0" align="center" style="text-align: center; color:#f09600; border: 1.5px solid #333333;"
|- border="0"
|- border="0"
-
! colspan="7" style="background:#009be1;" | [https://2010.igem.org/Team:Heidelberg/Notebook/Mouse_Infection/August<font color="white">August</font>]
+
! colspan="7" style="background:#78b41e;" | [https://2010.igem.org/Team:Heidelberg/Notebook/Mouse_Infection/October<font color="white">October</font>]
-
|- style="background:#009be1; color:white"
+
|- style="background:#78b41e; color:white"
|width="20pt"|'''M'''||width="20pt"|'''T'''||width="20pt"|'''W'''||width="20pt"|'''T'''||width="20pt"|'''F'''||width="20pt"|'''S'''||width="20pt"|'''S'''
|width="20pt"|'''M'''||width="20pt"|'''T'''||width="20pt"|'''W'''||width="20pt"|'''T'''||width="20pt"|'''F'''||width="20pt"|'''S'''||width="20pt"|'''S'''
-
|-  
+
|- style="background:#f2f2f2; color:#78b41e"
-
|colspan="6"| ||'''1'''
+
|colspan="4"| ||'''[https://2010.igem.org/Team:Heidelberg/Notebook/Mouse_Infection/October#01.2F10.2F2010 1]'''||'''[https://2010.igem.org/Team:Heidelberg/Notebook/Mouse_Infection/October#02.2F10.2F2010 2]'''||'''[https://2010.igem.org/Team:Heidelberg/Notebook/Mouse_Infection/October#03.2F10.2F2010 3]'''
-
|-
+
|- style="background:#f2f2f2; color:#78b41e"
-
|'''2'''||'''3'''||'''4'''||'''5'''||'''6'''||'''7'''||'''8'''
+
|'''[https://2010.igem.org/Team:Heidelberg/Notebook/Mouse_Infection/October#04.2F10.2F2010 4]'''||'''[https://2010.igem.org/Team:Heidelberg/Notebook/Mouse_Infection/October#05.2F10.2F2010 5]'''
-
|-
+
|'''[https://2010.igem.org/Team:Heidelberg/Notebook/Mouse_Infection/October#06.2F10.2F2010 6]'''||'''[https://2010.igem.org/Team:Heidelberg/Notebook/Mouse_Infection/October#07.2F10.2F2010 7]'''||'''[https://2010.igem.org/Team:Heidelberg/Notebook/Mouse_Infection/October#08.2F10.2F2010 8]'''||'''[https://2010.igem.org/Team:Heidelberg/Notebook/Mouse_Infection/October#09.2F10.2F2010 9]'''||'''[https://2010.igem.org/Team:Heidelberg/Notebook/Mouse_Infection/October#10.2F10.2F2010 10]'''
-
|'''9'''||'''10'''||'''11'''||'''12'''||'''13'''||'''14'''||'''15'''
+
|- style="background:#f2f2f2; color:#78b41e"
-
|-
+
|'''[https://2010.igem.org/Team:Heidelberg/Notebook/Mouse_Infection/October#11.2F10.2F2010 11]'''||'''[https://2010.igem.org/Team:Heidelberg/Notebook/Mouse_Infection/October#12.2F10.2F2010 12]'''|'''[https://2010.igem.org/Team:Heidelberg/Notebook/Mouse_Infection/October#13.2F10.2F2010 13]'''||'''[https://2010.igem.org/Team:Heidelberg/Notebook/Mouse_Infection/October#14.2F10.2F2010 14]'''||'''[https://2010.igem.org/Team:Heidelberg/Notebook/Mouse_Infection/October#15.2F10.2F2010 15]'''||'''[https://2010.igem.org/Team:Heidelberg/Notebook/Mouse_Infection/October#16.2F10.2F2010 16]'''||'''[https://2010.igem.org/Team:Heidelberg/Notebook/Mouse_Infection/October#17.2F10.2F2010 17]'''||'''[https://2010.igem.org/Team:Heidelberg/Notebook/Mouse_Infection/October#18.2F10.2F2010 18]'''
-
|'''16'''||'''17'''||'''18'''||'''19'''||'''20'''||'''[[Igem2010/Main/synthetic_miR_Kit/August#21/08/2010|21]]'''||'''[[Igem2010/Main/synthetic_miR_Kit/August#22/08/2010|22]]'''
+
|- style="background:#f2f2f2; color:#78b41e"
-
|-
+
|'''[https://2010.igem.org/Team:Heidelberg/Notebook/Mouse_Infection/October#19.2F10.2F2010 19]'''||'''[https://2010.igem.org/Team:Heidelberg/Notebook/Mouse_Infection/October#20.2F10.2F2010 20]'''||'''[https://2010.igem.org/Team:Heidelberg/Notebook/Mouse_Infection/October#21.2F10.2F2010 21]'''||'''[https://2010.igem.org/Team:Heidelberg/Notebook/Mouse_Infection/October#22.2F10.2F2010 22]'''||'''[https://2010.igem.org/Team:Heidelberg/Notebook/Mouse_Infection/October#23.2F10.2F2010 23]'''||'''[https://2010.igem.org/Team:Heidelberg/Notebook/Mouse_Infection/October#24.2F10.2F2010 24]'''||'''[https://2010.igem.org/Team:Heidelberg/Notebook/Mouse_Infection/October#25.2F10.2F2010 25]'''
-
|'''[[Igem2010/Main/synthetic_miR_Kit/August#23/08/2010|23]]'''||'''[[Igem2010/Main/synthetic_miR_Kit/August#21/08/2010|24]]'''||'''[[Igem2010/Main/synthetic_miR_Kit/August#21/08/2010|25]]'''||'''[[Igem2010/Main/synthetic_miR_Kit/August#26/08/2010|26]]'''||'''[[Igem2010/Main/synthetic_miR_Kit/August#27/08/2010|27]]'''||'''[[Igem2010/Main/synthetic_miR_Kit/August#28/08/2010|28]]'''||'''[[Igem2010/Main/synthetic_miR_Kit/August#29/08/2010|29]]'''
+
|- style="background:#f2f2f2; color:#78b41e"
-
|-
+
|'''[https://2010.igem.org/Team:Heidelberg/Notebook/Mouse_Infection/October#26.2F10.2F2010 26]'''||'''[https://2010.igem.org/Team:Heidelberg/Notebook/Mouse_Infection/October#27.2F10.2F2010 27]'''||'''[https://2010.igem.org/Team:Heidelberg/Notebook/Mouse_Infection/October#28.2F10.2F2010 28]'''||'''[https://2010.igem.org/Team:Heidelberg/Notebook/Mouse_Infection/October#29.2F10.2F2010 29]'''||'''[https://2010.igem.org/Team:Heidelberg/Notebook/Mouse_Infection/October#22.2F30.2F2010 30]'''||colspan="3"|
-
|'''[[Igem2010/Main/synthetic_miR_Kit/August#30/08/2010|30]]'''||'''[[Igem2010/Main/synthetic_miR_Kit/August#31/08/2010|31]]'''||colspan="5"|
+
|- style="background:#f2f2f2; color:#78b41e"
 +
|colspan="7"|
 +
<span style="color:#ffffff">-</span>
|}
|}
-
{| cellpadding="5" cellspacing="0" style="text-align: center; color:#f3d675; border: 3px solid #4e93a4;"
 
-
|- border="0"
 
-
! colspan="7" style="background:#4e93a4;" | [https://2010.igem.org/Team:Heidelberg/Notebook/Mouse_Infection/September<font color="#ffecba">September</font>]
 
-
|- style="background:#4e93a4; color:white"
 
-
|width="20pt"|'''M'''||width="20pt"|'''T'''||width="20pt"|'''W'''||width="20pt"|'''T'''||width="20pt"|'''F'''||width="20pt"|'''S'''||width="20pt"|'''S'''
 
-
|-
 
-
|colspan="2"| ||'''[[Igem2010/Main/synthetic_miR_Kit/September#01/09/2010|1]]'''||'''[[Igem2010/Main/synthetic_miR_Kit/September#02/09/2010|2]]'''||'''[[Igem2010/Main/synthetic_miR_Kit/September#03/09/2010|3]]'''||'''[[Igem2010/Main/synthetic_miR_Kit/September#04/09/2010|4]]'''||'''[[Igem2010/Main/synthetic_miR_Kit/September#05/09/2010|5]]'''
 
-
|-
 
-
|'''[[Igem2010/Main/synthetic_miR_Kit/September#06/09/2010|6]]'''||'''[[Igem2010/Main/synthetic_miR_Kit/September#07/09/2010|7]]'''||'''[[Igem2010/Main/synthetic_miR_Kit/September#08/09/2010|8]]'''||'''[[Igem2010/Main/synthetic_miR_Kit/September#09/09/2010|9]]'''||'''[[Igem2010/Main/synthetic_miR_Kit/September#10/09/2010|10]]'''||'''[[Igem2010/Main/synthetic_miR_Kit/September#11/09/2010|11]]'''||'''[[Igem2010/Main/synthetic_miR_Kit/September#12/09/2010|12]]'''
 
-
|-
 
-
|'''[[Igem2010/Main/synthetic_miR_Kit/September#13/09/2010|13]]'''||'''[[Igem2010/Main/synthetic_miR_Kit/September#14/09/2010|14]]'''||'''[[Igem2010/Main/synthetic_miR_Kit/September#15/09/2010|15]]'''||'''[[Igem2010/Main/synthetic_miR_Kit/September#16/09/2010|16]]'''||'''[[Igem2010/Main/synthetic_miR_Kit/September#17/09/2010|17]]'''||'''[[Igem2010/Main/synthetic_miR_Kit/September#18/09/2010|18]]'''||'''[[Igem2010/Main/synthetic_miR_Kit/September#19/09/2010|19]]'''
 
-
|-
 
-
|'''[[Igem2010/Main/synthetic_miR_Kit/September#20/09/2010|20]]'''||'''[[Igem2010/Main/synthetic_miR_Kit/September#21/09/2010|21]]'''||'''[[Igem2010/Main/synthetic_miR_Kit/September#22/09/2010|22]]'''||'''[[Igem2010/Main/synthetic_miR_Kit/September#23/09/2010|23]]'''||'''[[Igem2010/Main/synthetic_miR_Kit/September#24/09/2010|24]]'''||'''[[Igem2010/Main/synthetic_miR_Kit/September#25/09/2010|25]]'''||'''[[Igem2010/Main/synthetic_miR_Kit/September#26/09/2010|26]]'''
 
-
|-
 
-
|'''[[Igem2010/Main/synthetic_miR_Kit/September#27/09/2010|27]]'''||'''[[Igem2010/Main/synthetic_miR_Kit/September#28/09/2010|28]]'''||'''[[Igem2010/Main/synthetic_miR_Kit/September#29/09/2010|29]]'''||'''[[Igem2010/Main/synthetic_miR_Kit/September#30/09/2010|30]]'''||colspan="3"|
 
-
|-
 
-
| colspan="7"| <span style="color:#ffffff">-</span>
 
-
|}
 
-
 
-
{| cellpadding="5" cellspacing="0" style="text-align: center; color:#f3d675; border: 3px solid #4e93a4;"
 
-
|- border="0"
 
-
! colspan="7" style="background:#4e93a4;" | [https://2010.igem.org/Igem2010/Main/Mouse_Infection/October<font color="#ffecba">October</font>]
 
-
|- style="background:#4e93a4; color:white"
 
-
|width="20pt"|'''M'''||width="20pt"|'''T'''||width="20pt"|'''W'''||width="20pt"|'''T'''||width="20pt"|'''F'''||width="20pt"|'''S'''||width="20pt"|'''S'''
 
-
|-
 
-
|colspan="4"| ||'''[[Igem2010/Main/synthetic_miR_Kit/October#01/10/2010|01]]'''||'''[[Igem2010/Main/synthetic_miR_Kit/October#02/10/2010|02]]'''||'''[[Igem2010/Main/synthetic_miR_Kit/October#03/10/2010|03]]'''
 
-
|-
 
-
|'''[[Igem2010/Main/synthetic_miR_Kit/October#04/10/2010|04]]'''||'''[[Igem2010/Main/synthetic_miR_Kit/October#05/10/2010|05]]'''||'''[[Igem2010/Main/synthetic_miR_Kit/October#06/10/2010|06]]'''||'''[[Igem2010/Main/synthetic_miR_Kit/October#07/10/2010|07]]'''||'''[[Igem2010/Main/synthetic_miR_Kit/October#08/10/2010|08]]'''||'''[[Igem2010/Main/synthetic_miR_Kit/October#09/10/2010|09]]'''||'''[[Igem2010/Main/synthetic_miR_Kit/October#10/10/2010|10]]'''
 
-
|-
 
-
|'''[[Igem2010/Main/synthetic_miR_Kit/October#11/10/2010|11]]'''||'''[[Igem2010/Main/synthetic_miR_Kit/October#12/10/2010|12]]'''||'''[[Igem2010/Main/synthetic_miR_Kit/October#13/10/2010|13]]'''||'''[[Igem2010/Main/synthetic_miR_Kit/October#14/10/2010|14]]'''||'''[[Igem2010/Main/synthetic_miR_Kit/October#15/10/2010|15]]'''||'''[[Igem2010/Main/synthetic_miR_Kit/October#16/10/2010|16]]'''||'''[[Igem2010/Main/synthetic_miR_Kit/October#17/10/2010|17]]'''
 
-
|-
 
-
|'''[[Igem2010/Main/synthetic_miR_Kit/October#18/10/2010|18]]'''||'''[[Igem2010/Main/synthetic_miR_Kit/October#19/10/2010|19]]'''||'''[[Igem2010/Main/synthetic_miR_Kit/October#20/10/2010|20]]'''||'''[[Igem2010/Main/synthetic_miR_Kit/October#21/10/2010|21]]'''||'''[[Igem2010/Main/synthetic_miR_Kit/October#22/10/2010|22]]'''||'''[[Igem2010/Main/synthetic_miR_Kit/October#23/10/2010|23]]'''||'''[[Igem2010/Main/synthetic_miR_Kit/October#24/10/2010|24]]'''
 
-
|-
 
-
|'''[[Igem2010/Main/synthetic_miR_Kit/October#25/10/2010|25]]'''||'''[[Igem2010/Main/synthetic_miR_Kit/October#26/10/2010|26]]'''||'''[[Igem2010/Main/synthetic_miR_Kit/October#27/10/2010|27]]'''||'''28'''||'''29'''||'''30'''||'''31'''
 
-
|-
 
-
| colspan="7"| <span style="color:#ffffff">-</span>
 
-
|}
 
{{:Team:Heidelberg/Side_Bottom}}
{{:Team:Heidelberg/Side_Bottom}}
__NOTOC__
__NOTOC__

Revision as of 01:33, 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/Team:Heidelberg/Notebook/Mouse_Infection/October"