Team:Heidelberg/Notebook/Mouse Infection

From 2010.igem.org

(Difference between revisions)
Line 2: Line 2:
{{:Team:Heidelberg/Pagetop|home}}
{{:Team:Heidelberg/Pagetop|home}}
-
=Mouse Infection=
+
= Mouse Infection =
-
=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==
+
{{:Team:Heidelberg/Pagemiddle}}
-
* 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
+
{| cellpadding="5" cellspacing="0" style="text-align: center; color:#009be1; border: 3px solid #009be1;"
-
* mice were injected the following:
+
|- border="0"
-
* 1) SV40-Luc2-miR122 perfect binding site in AAV8 capsid
+
! colspan="7" style="background:#009be1;" | [https://2010.igem.org/Team:Heidelberg/Notebook/Mouse_Infection/August<font color="white">August</font>]
-
* 2) SV40-Luc2 in our shuffeled capsid
+
|- style="background:#009be1; color:white"
-
* 3) SV40-Luc2 in AAV8 capsid
+
|width="20pt"|'''M'''||width="20pt"|'''T'''||width="20pt"|'''W'''||width="20pt"|'''T'''||width="20pt"|'''F'''||width="20pt"|'''S'''||width="20pt"|'''S'''
-
* 4) SV40-Luc2-miRhaat perfect binding site in AAV8 capsid
+
|-
-
* 5) SV40-Luc2-miRhaat imperfect binding site (9-12) in AAV8 capsid
+
|colspan="6"| ||'''1'''
-
* 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
+
|'''2'''||'''3'''||'''4'''||'''5'''||'''6'''||'''7'''||'''8'''
-
==25/10/2010==
+
|-
-
*plate 40 15 cm dishes with 5.5*10^6 cells per dish
+
|'''9'''||'''10'''||'''11'''||'''12'''||'''13'''||'''14'''||'''15'''
-
==26/10/2010==
+
|-
-
*transfect all 40 plates using PEI transfection buffer and the following set-up
+
|'''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]]'''
-
==27/10/2010==
+
|-
-
* bioluminescence measurements on mice from the first injection round
+
|'''[[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]]'''
 +
|-
 +
|'''[[Igem2010/Main/synthetic_miR_Kit/August#30/08/2010|30]]'''||'''[[Igem2010/Main/synthetic_miR_Kit/August#31/08/2010|31]]'''||colspan="5"|
 +
|}
 +
{| 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>
 +
|}
-
{{:Team:Heidelberg/Pagemiddle}}
+
{| 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/Bottom}}
{{:Team:Heidelberg/Bottom}}

Revision as of 12:17, 24 October 2010

Mouse Infection

August
MTWTFSS
1
2345678
9101112131415
16171819202122
23242526272829
3031
September
MTWTFSS
12345
6789101112
13141516171819
20212223242526
27282930
-
October
MTWTFSS
010203
04050607080910
11121314151617
18192021222324
25262728293031
-

Retrieved from "http://2010.igem.org/Team:Heidelberg/Notebook/Mouse_Infection"