Team:Heidelberg/Notebook/Measurement Standard

From 2010.igem.org

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=Measurement Standard=
=Measurement Standard=
 +
'''[[Igem2010/Main|⇐ Main Page]]'''
 +
 +
'''[[Igem2010/Main/Measurements/Notebook|⇐ Measurements]]'''
 +
 +
----
 +
 +
=08/08/2010=
 +
 +
seeding cells for test measurements - 96 well plate for plate-reader and FACS
 +
 +
*cells were grown in DMEM 10%FBS with phenol red
 +
*washed with PBS
 +
*trypsinised (2 ml trypsin)
 +
*5ml of OptiMEM media (no FBS) added
 +
*counted cells: HeLa 3.8*10^6 cells/ml, HEK 3.3*10^6 cells/ml, HEK T-Rex 1.5*10^6 cells/ml, Huh7 0.8*10^6 cells/ml
 +
*seeded 5000 and 2500 cells/well as on the scheme [[96well plate 080810.jpg]]
 +
*media: DMEM +L-Glu +PenStrep +10%FBS, OptiMEM +PenStrep, OptiMEM +PenStrep +2%FBS
 +
*in all wells where T-Rex cells were seeded zeocin and blasticin were added, in wells with Huh7 cells - non essential amino acids
 +
 +
-> cells did not adhere well, coat plate with poly-L-lysine and repeat
 +
 +
----
 +
 +
=09/08/2010=
 +
 +
seeding and transfecting cells for microscopy test measurements
 +
 +
* add 0.6ul FuGENE reagent to 20ul of OptiMEM
 +
* mix and incubate 5' at RT
 +
* add 0.2ug DNA
 +
* mix and incubate 15' at RT
 +
* add DNA-FuGENE solution to 10 000 cells (400ul)
 +
* mix and incubate 10' 37degC 300rpm (shaker for 15ml falcon tubes is in the cell culture room at 3rd floor)
 +
* transfere cells to the plate
 +
* grow 48h
 +
 +
 +
{| class="wikitable" style="border:solid 1px #AAAAAA; border-collapse:collapse;  background-color:#F9F9F9; font-size:95%; empty-cells:show;"
 +
| style="text-align:right" | HeLa, EGPF || HeLa, EBFP2 || HeLa, EGPF and EBFP2 ||  HeLa, EGPF and EBFP2 1:10
 +
|-
 +
| style="text-align:right" | HEK T-REx, EGFP || HEK T-REx, EBFP2 || HEK T-REx, EGPF and EBFP2 || HEK T-REx, EGPF and EBFP2 1:10
 +
|}
 +
 +
----
 +
 +
=10/08/2010=
 +
 +
coating plates with poly-L-lysine
 +
* add 15ul of poly-L-lysine solution to each well (make sure whole surface is covered with solution)
 +
* leave for 30' in the incubator
 +
* remove poly-L-lysine solution
 +
* wash once with PBS
 +
 +
new 96-well plate was prepared (wells 2500 cells Huh7 are contaminated with HeLa) like previously
 +
 +
----
 +
 +
=25/08/2010=
 +
 +
seeding cells for pilot FACS and Tecan measurements (96-well plate)
 +
 +
----
 +
 +
=26/08/2010=
 +
 +
transfection - 96-well plate
 +
 +
----
 +
 +
=27/08/2010=
 +
 +
pilot FACS and Tecan measurements (96-well plate)
 +
 +
 +
'''[[Igem2010/Main/Measurements/Notebook/September|⇓ September]]'''
 +
 +
'''[[Igem2010/Main|⇐ Main Page]]'''
 +
 +
'''[[Igem2010/Main/Measurements/Notebook|⇐ Measurements]]'''
 +
 +
'''[[Igem2010/Main/Measurements/Notebook/August|⇑ August]]'''
 +
 +
----
 +
 +
=02/09/2010=
 +
*seeding 1 96-well plate with all different cell lines we have (HelaP4, Hek293T and Huh7) for testing fluorescent measurement:
 +
** purpose is to set up plate reader (TECAN), FACS and microscope and test whether it is possible to distinguish between GFP and BFP expression
 +
** problem: forgot to coat the plate, therefore cells did not attach
 +
 +
=03/09/2010=
 +
[[Image:HeLa_pilot_GFP_BFP2.jpg|thumb|500px‎|FACS pilot experiment of EGFP and EBFP2 expression in Hela P4 cells]]
 +
[[Image:Huh7_pilot_GFP_BFP2.jpg|thumb|300px|FACS pilot experiment of EGFP and EBFP2 expression in Hek T-Rex cells]][[Image:HEK_pilot_GFP_BFP2.jpg|thumb|300px|Tecan pilot experiment of EBFP2 and EGFP expression with in Hela P4 cells‎‎]]
 +
[[Image:HEK_TREx_pilot_GFP_BFP2.jpg|thumb|300px|Tecan pilot experiment of EBFP2 and EGFP expression with in Hek T-Rex cells‎‎]]
 +
*transfection of cells for fluorescent measurement (EGFP and EBFP2):
 +
 +
*set-up of the plate:
 +
** non-transfected cell line
 +
** each cell line transfected with EGFP control plasmid
 +
** each cell line transfected with EBFP2 control plasmid
 +
** each cell line double-transfected with EGFP and EBFP2 control plasmids
 +
** Results:
 +
*** too little cells have been plated
 +
*** Huh7 cells are hard to transfect
 +
*** expression of EBFP2 is much lower than expression of EGFP
 +
<br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br>
 +
=04/09/2010=
 +
 +
*seeding 1 96-well plate with all different cell lines we have (HelaP4, Hek293T and Huh7) for testing fluorescent measurement:
 +
** purpose is to set up plate reader (TECAN), FACS and microscope and test whether it is possible to distinguish between GFP and BFP expression
 +
=05/09/2010=
 +
*transfection of cells for fluorescent measurement (EGFP and EBFP2):
 +
*set-up of the plate:
 +
** non-transfected cell line
 +
** each cell line transfected with EGFP control plasmid
 +
** each cell line transfected with EBFP2 control plasmid
 +
** each cell line double-transfected with EGFP and EBFP2 control plasmids
 +
 +
=06/09/2010=
 +
*TECAN and FACS pilot measurements of cells transfected with EGFP and EBFP2
 +
 +
=07/09/2010=
 +
*seeding and transfection of HeLa and HEK cells for pilot microscopy measurements:
 +
 +
 +
=09/09/2010=
 +
*pilot microscopy measurements:
 +
**
 +
 +
=20/09/2010=
 +
*seeding 2 96-well plates with 5000 cells each well:
 +
**test whether tuning construct is properly expressing firefly and renilla luciferase and thereby testing whether every part is functional
 +
 +
=21/09/2010=
 +
*transfection of both 96-well plates with K1-K8:
 +
** transfect 50ng of each construct
 +
** check for promoter strength and functionality of the constructs
 +
<br><br><br><br><br><br><br><br><br>
 +
=22/09/2010=
 +
[[Image:22092010promoters.jpg|thumb|400px|right|relative expression units(REU) of firefly luciferase compared to renilla luciferase of the different tuning consstructs without any binding sites for the expressed shRNA10]]
 +
[[Image:22092010promoters2.jpg|thumb|400px|left|comparison of REU of firefly to renilla luciferase in the different cell lines]]
 +
*dual luciferase assay on the 2 96-well plates:
 +
** all constructs are functional as firefly and renilla luciferase are expressed
 +
** transfection of construct into Hek t-Rex cells reveals that the CMVTetO2 (construct K4) is also functional as the expression of firefly to renilla counts is nearly zero
 +
<br><br><br><br><br>
 +
 +
=23/09/2010=
 +
dual luciferase assay
 +
 +
[[Image:23092010promoters.jpg|thumb|500px|right]]
 +
[[Image:23092010promoters2.jpg|thumb|500px|right]]
 +
 +
=1/10/2010=
 +
seed two 96-well plates with HEK cells 5000 cells/well
 +
 +
=2/10/2010=
 +
transfection
 +
=11/10/2010=
 +
*transfection of 24-well plates for 50 clones of shuffled capsid
 +
*coat 6-well plates with collagen for primary hepatocytes
 +
*measurements of miMeasure
 +
 +
=12/10/2010=
 +
* seed 1 96-well plate for transfection with miMeasure
 +
** 6 lines Huh7
 +
** 6 lines HepG2
 +
*Dual Luciferase assay for ??
 +
*seed 24-well plates with Huh7 for second selection round
 +
=13/10/2010=
 +
* seeding of primary hepatocytes for first selection round of capsid library on a 6-well plate
 +
* seeding of primary hepatocytes for selection round of 50 clones on 2 24-well plates
 +
 +
=14/10/2010=
 +
* transfection of 1 96-well plate with off targeting constructs:
 +
** M23-M29 with or without co-transfection of miR122 of miRsag as a control
 +
* transfection of tuning construct K4 with or without co-transfection of miRsAg and miRhaat as a control
 +
=15/10/2010=
 +
* Dual luciferase assay on tuning construct with co-transfection of shRNA miRsag and miR122
 +
* Dual luciferase assay of off target constructs
 +
* seeding of :
 +
** 1 96-well plate of half Huh7 and half HepG2
 +
** 1 plate for testing nuclei staining with Hela, HepG2 and Huh7
 +
* prepare 3 bottles of media
 +
* organize 500 Million Hek293T cells :)
 +
*change media from primary hepatocytes
 +
 +
=15/10/2010=
 +
* plate 1 96-well plate with half of the wells with Huh7 and other half with HepG2
 +
* plate half of a 96-well plate with Hela, HepG2 and Huh7 for testing nuclei staining for TECAN
 +
=16/10/2010=
 +
*transfect 96-well plate of liver cell lines with miMeasure with perfect, imperfect 9-12 and imperfect 9-22 binding site
 +
* transfect test staining plate with miMeasure in different concentrations:
 +
** 10ng of miMeasure in 3 wells of each cell line
 +
** 30ng of miMeasure in 3 wells of each cell line
 +
* plate 8 96-well plates with Hela cells
 +
* plate 1 96-well plate with half Huh7 and HepG2
 +
=17/10/2010=
 +
* transfect 3 96-well plates with tuning construct for in vivo:
 +
** tuning construct M1-M10 without any synthetic miRNA
 +
** co-transfection of tuning construct M1-M10 with synthetic miRhaat
 +
** co-transfection of tuning construct M1-M10 with synthetic miRSag
 +
* transfect 3 96-well plates with miMeasure with different binding sites against miRsag:
 +
** miMeasure M12-M22 without any synthetic miRNA
 +
** co-transfection of miMeasure M12-M22 with synthetic miRhaat
 +
** co-transfection of miMeasure M12-M22 with synthetic miRSag
 +
* coat 1 6-well plate with collagene for primary hepatocytes
 +
=18/10/2010=
 +
* measure 3 96-well plates with tuning construct for in vivo by Dual luciferase assay
 +
** measurement did not work, no luciferase activity could be measured
 +
* measure 1 96-well plate of miMeasure with binding sites for miR122
 +
** measurement did not work, maybe too little DNA transfected in the cells (10ng) as Huh7 are not easily transfected
 +
* measure plate for testing nuclei staining
 +
** measurement did not work
 +
* coat plates with collagene (2* 6-well plate) for primary hepatocytes
 +
* plate 1 96-well plate with Huh7 and HepG2
 +
* plate 24 well plate with Hela cells for haat Elisa
 +
=19/10/2010=
 +
* transfect miMeasure into Huh7 and HepG2 on 96-well plate again with binding sites of miR122:
 +
** perfect binding site
 +
** imperfect binding sites (randomized 9-12)
 +
** imperfect binding sites (randomized 9-22)
 +
*transfect haat cDNA constructs into Hela cells on 24-well plates
 +
 +
*measurement M12-M19
 +
**TECAN
 +
**did not work, because cells were clustered
 +
**Confocal
 +
**cells from the 96 well plate were pooled together and single pictures were made.
 +
 +
[[Image:M12-M22_confocalS.pdf]]
 +
 +
*who deleted what I wrote here??? hallooo?? hmm I guess I just didn't save it. damn.
 +
 +
=23/10/2010=
 +
 +
* measurement of Hela and Hek Cells transfected with miMeasure M12-M22. Four conditions were transfected with four replicates each: a = not cotransfected, b = cotransfected with miRsAg on pcDNA5, c = cotransfected with empty pcDNA5 (CMV promoter), d = cotransfected with pcDNA5 containing different shRNA (shRNA3 from Elena).
 +
** because of a contamination, not all replicates could be considered. Plates were screened for positive transfections
 +
** cells were trypsinated (30µl Trypsin) for 10min and then resuspended with 170µl PBS/BSA. Replicates for each condition were pooled into 24 well plates.
 +
**200µl of each was transferred to a 96 well plate for FACS measurement.
 +
**100µl of each was used for confocal analysis
 +
 +
 +
{{:Team:Heidelberg/Pagemiddle}}
{{:Team:Heidelberg/Pagemiddle}}
 +
how do I get a calendar in here??? anyone??
 +
'''[[Igem2010/Main|&lArr; Main Page]]'''
 +
 +
 +
{| cellspacing="10" style="color:#c0c0c0"
 +
|-
 +
|
 +
{| cellpadding="5" cellspacing="0" style="text-align: center; color:#f3d675; border: 3px solid #4e93a4;"
 +
|- border="0"
 +
! colspan="7" style="background:#4e93a4;" | [[Igem2010/Main/Measurements/Notebook/July|<font color="#ffecba">July</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="3"| ||'''1'''||'''2'''||'''3'''||'''4'''
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|-
 +
|'''5'''||'''6'''||'''7'''||'''8'''||'''9'''||'''10'''||'''11'''
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|-
 +
|'''12'''||'''13'''||'''14'''||'''15'''||'''16'''||'''17'''||'''18'''
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|-
 +
|'''19'''||'''20'''||'''21'''||'''22'''||'''23'''||'''24'''||'''25'''
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|-
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|'''26'''||'''27'''||'''28'''||'''29'''||'''30'''||'''31'''||
 +
|-
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| 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;" | [[Igem2010/Main/Measurements/Notebook/August|<font color="#ffecba">August</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="6"| ||'''1'''
 +
|-
 +
|'''2'''||'''3'''||'''4'''||'''5'''||'''6'''||'''7'''||'''[[Igem2010/Main/Measurements/Notebook/August#08/08/2010|8]]'''
 +
|-
 +
|'''[[Igem2010/Main/Measurements/Notebook/August#09/08/2010/|9]]'''||'''[[Igem2010/Main/Measurements/Notebook/August#10/08/2010|10]]'''||'''11'''||'''12'''||'''13'''||'''14'''||'''15'''
 +
|-
 +
|'''16'''||'''17'''||'''18'''||'''19'''||'''20'''||'''21'''||'''22'''
 +
|-
 +
|'''23'''||'''24'''||'''[[Igem2010/Main/Measurements/Notebook/August#25/08/2010|25]]'''||'''[[Igem2010/Main/Measurements/Notebook/August#26/08/2010|26]]'''||'''[[Igem2010/Main/Measurements/Notebook/August#27/08/2010|27]]'''||'''28'''||'''29'''
 +
|-
 +
|'''30'''||'''31'''||colspan="5"|
 +
|}
 +
|
 +
{| cellpadding="5" cellspacing="0" style="text-align: center; color:#f3d675; border: 3px solid #4e93a4;"
 +
|- border="0"
 +
! colspan="7" style="background:#4e93a4;" | [[Igem2010/Main/Measurements/Notebook/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"| ||'''1'''||'''[[Igem2010/Main/Measurements/Notebook/September#02/09/2010|2]]'''||'''[[Igem2010/Main/Measurements/Notebook/September#03/09/2010|3]]'''||'''4'''||'''5'''
 +
|-
 +
|'''[[Igem2010/Main/Measurements/Notebook/September#06/09/2010|6]]'''||'''[[Igem2010/Main/Measurements/Notebook/September#07/09/2010|7]]'''||'''8'''||'''[[Igem2010/Main/Measurements/Notebook/September#09/09/2010|9]]'''||'''10'''||'''11'''||'''12'''
 +
|-
 +
|'''13'''||'''14'''||'''15'''||'''16'''||'''17'''||'''18'''||'''19'''
 +
|-
 +
|'''[[Igem2010/Main/Measurements/Notebook/September#20/09/2010|20]]'''||'''[[Igem2010/Main/Measurements/Notebook/September#21/09/2010|21]]'''||'''[[Igem2010/Main/Measurements/Notebook/September#22/09/2010|22]]'''||'''[[Igem2010/Main/Measurements/Notebook/September#23/09/2010|23]]'''||'''24'''||'''25'''||'''26'''
 +
|-
 +
|'''27'''||'''28'''||'''29'''||'''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;" | [[Igem2010/Main/Measurements/Notebook/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/Measurements/Notebook/October#1/09/2010|1]]'''||'''[[Igem2010/Main/Measurements/Notebook/October#2/09/2010|2]]'''||'''3'''
 +
|-
 +
|'''4'''||'''5'''||'''6'''||'''7'''||'''8'''||'''9'''||'''10'''
 +
|-
 +
|'''11'''||'''12'''||'''13'''||'''14'''||'''15'''||'''16'''||'''17'''
 +
|-
 +
|'''18'''||'''19'''||'''20'''||'''21'''||'''22'''||'''23'''||'''24'''
 +
|-
 +
|'''25'''||'''26'''||'''27'''||'''28'''||'''29'''||'''30'''||'''31'''
 +
|-
 +
| colspan="7"| <span style="color:#ffffff">-</span>
 +
|}
 +
|}
 +
{{:Team:Heidelberg/Bottom}}
{{:Team:Heidelberg/Bottom}}

Latest revision as of 09:49, 25 October 2010

Contents

Measurement Standard

⇐ Main Page

⇐ Measurements

08/08/2010

seeding cells for test measurements - 96 well plate for plate-reader and FACS

  • cells were grown in DMEM 10%FBS with phenol red
  • washed with PBS
  • trypsinised (2 ml trypsin)
  • 5ml of OptiMEM media (no FBS) added
  • counted cells: HeLa 3.8*10^6 cells/ml, HEK 3.3*10^6 cells/ml, HEK T-Rex 1.5*10^6 cells/ml, Huh7 0.8*10^6 cells/ml
  • seeded 5000 and 2500 cells/well as on the scheme 96well plate 080810.jpg
  • media: DMEM +L-Glu +PenStrep +10%FBS, OptiMEM +PenStrep, OptiMEM +PenStrep +2%FBS
  • in all wells where T-Rex cells were seeded zeocin and blasticin were added, in wells with Huh7 cells - non essential amino acids

-> cells did not adhere well, coat plate with poly-L-lysine and repeat

09/08/2010

seeding and transfecting cells for microscopy test measurements

  • add 0.6ul FuGENE reagent to 20ul of OptiMEM
  • mix and incubate 5' at RT
  • add 0.2ug DNA
  • mix and incubate 15' at RT
  • add DNA-FuGENE solution to 10 000 cells (400ul)
  • mix and incubate 10' 37degC 300rpm (shaker for 15ml falcon tubes is in the cell culture room at 3rd floor)
  • transfere cells to the plate
  • grow 48h


HeLa, EGPF HeLa, EBFP2 HeLa, EGPF and EBFP2 HeLa, EGPF and EBFP2 1:10
HEK T-REx, EGFP HEK T-REx, EBFP2 HEK T-REx, EGPF and EBFP2 HEK T-REx, EGPF and EBFP2 1:10

10/08/2010

coating plates with poly-L-lysine

  • add 15ul of poly-L-lysine solution to each well (make sure whole surface is covered with solution)
  • leave for 30' in the incubator
  • remove poly-L-lysine solution
  • wash once with PBS

new 96-well plate was prepared (wells 2500 cells Huh7 are contaminated with HeLa) like previously

25/08/2010

seeding cells for pilot FACS and Tecan measurements (96-well plate)

26/08/2010

transfection - 96-well plate

27/08/2010

pilot FACS and Tecan measurements (96-well plate)


⇓ September

⇐ Main Page

⇐ Measurements

⇑ August

02/09/2010

  • seeding 1 96-well plate with all different cell lines we have (HelaP4, Hek293T and Huh7) for testing fluorescent measurement:
    • purpose is to set up plate reader (TECAN), FACS and microscope and test whether it is possible to distinguish between GFP and BFP expression
    • problem: forgot to coat the plate, therefore cells did not attach

03/09/2010

File:HeLa pilot GFP BFP2.jpg
FACS pilot experiment of EGFP and EBFP2 expression in Hela P4 cells
File:Huh7 pilot GFP BFP2.jpg
FACS pilot experiment of EGFP and EBFP2 expression in Hek T-Rex cells
File:HEK pilot GFP BFP2.jpg
Tecan pilot experiment of EBFP2 and EGFP expression with in Hela P4 cells‎‎
File:HEK TREx pilot GFP BFP2.jpg
Tecan pilot experiment of EBFP2 and EGFP expression with in Hek T-Rex cells‎‎
  • transfection of cells for fluorescent measurement (EGFP and EBFP2):
  • set-up of the plate:
    • non-transfected cell line
    • each cell line transfected with EGFP control plasmid
    • each cell line transfected with EBFP2 control plasmid
    • each cell line double-transfected with EGFP and EBFP2 control plasmids
    • Results:
      • too little cells have been plated
      • Huh7 cells are hard to transfect
      • expression of EBFP2 is much lower than expression of EGFP





































04/09/2010

  • seeding 1 96-well plate with all different cell lines we have (HelaP4, Hek293T and Huh7) for testing fluorescent measurement:
    • purpose is to set up plate reader (TECAN), FACS and microscope and test whether it is possible to distinguish between GFP and BFP expression

05/09/2010

  • transfection of cells for fluorescent measurement (EGFP and EBFP2):
  • set-up of the plate:
    • non-transfected cell line
    • each cell line transfected with EGFP control plasmid
    • each cell line transfected with EBFP2 control plasmid
    • each cell line double-transfected with EGFP and EBFP2 control plasmids

06/09/2010

  • TECAN and FACS pilot measurements of cells transfected with EGFP and EBFP2

07/09/2010

  • seeding and transfection of HeLa and HEK cells for pilot microscopy measurements:


09/09/2010

  • pilot microscopy measurements:

20/09/2010

  • seeding 2 96-well plates with 5000 cells each well:
    • test whether tuning construct is properly expressing firefly and renilla luciferase and thereby testing whether every part is functional

21/09/2010

  • transfection of both 96-well plates with K1-K8:
    • transfect 50ng of each construct
    • check for promoter strength and functionality of the constructs










22/09/2010

File:22092010promoters.jpg
relative expression units(REU) of firefly luciferase compared to renilla luciferase of the different tuning consstructs without any binding sites for the expressed shRNA10
File:22092010promoters2.jpg
comparison of REU of firefly to renilla luciferase in the different cell lines
  • dual luciferase assay on the 2 96-well plates:
    • all constructs are functional as firefly and renilla luciferase are expressed
    • transfection of construct into Hek t-Rex cells reveals that the CMVTetO2 (construct K4) is also functional as the expression of firefly to renilla counts is nearly zero






23/09/2010

dual luciferase assay

File:23092010promoters.jpg
500px
File:23092010promoters2.jpg
500px

1/10/2010

seed two 96-well plates with HEK cells 5000 cells/well

2/10/2010

transfection

11/10/2010

  • transfection of 24-well plates for 50 clones of shuffled capsid
  • coat 6-well plates with collagen for primary hepatocytes
  • measurements of miMeasure

12/10/2010

  • seed 1 96-well plate for transfection with miMeasure
    • 6 lines Huh7
    • 6 lines HepG2
  • Dual Luciferase assay for ??
  • seed 24-well plates with Huh7 for second selection round

13/10/2010

  • seeding of primary hepatocytes for first selection round of capsid library on a 6-well plate
  • seeding of primary hepatocytes for selection round of 50 clones on 2 24-well plates

14/10/2010

  • transfection of 1 96-well plate with off targeting constructs:
    • M23-M29 with or without co-transfection of miR122 of miRsag as a control
  • transfection of tuning construct K4 with or without co-transfection of miRsAg and miRhaat as a control

15/10/2010

  • Dual luciferase assay on tuning construct with co-transfection of shRNA miRsag and miR122
  • Dual luciferase assay of off target constructs
  • seeding of :
    • 1 96-well plate of half Huh7 and half HepG2
    • 1 plate for testing nuclei staining with Hela, HepG2 and Huh7
  • prepare 3 bottles of media
  • organize 500 Million Hek293T cells :)
  • change media from primary hepatocytes

15/10/2010

  • plate 1 96-well plate with half of the wells with Huh7 and other half with HepG2
  • plate half of a 96-well plate with Hela, HepG2 and Huh7 for testing nuclei staining for TECAN

16/10/2010

  • transfect 96-well plate of liver cell lines with miMeasure with perfect, imperfect 9-12 and imperfect 9-22 binding site
  • transfect test staining plate with miMeasure in different concentrations:
    • 10ng of miMeasure in 3 wells of each cell line
    • 30ng of miMeasure in 3 wells of each cell line
  • plate 8 96-well plates with Hela cells
  • plate 1 96-well plate with half Huh7 and HepG2

17/10/2010

  • transfect 3 96-well plates with tuning construct for in vivo:
    • tuning construct M1-M10 without any synthetic miRNA
    • co-transfection of tuning construct M1-M10 with synthetic miRhaat
    • co-transfection of tuning construct M1-M10 with synthetic miRSag
  • transfect 3 96-well plates with miMeasure with different binding sites against miRsag:
    • miMeasure M12-M22 without any synthetic miRNA
    • co-transfection of miMeasure M12-M22 with synthetic miRhaat
    • co-transfection of miMeasure M12-M22 with synthetic miRSag
  • coat 1 6-well plate with collagene for primary hepatocytes

18/10/2010

  • measure 3 96-well plates with tuning construct for in vivo by Dual luciferase assay
    • measurement did not work, no luciferase activity could be measured
  • measure 1 96-well plate of miMeasure with binding sites for miR122
    • measurement did not work, maybe too little DNA transfected in the cells (10ng) as Huh7 are not easily transfected
  • measure plate for testing nuclei staining
    • measurement did not work
  • coat plates with collagene (2* 6-well plate) for primary hepatocytes
  • plate 1 96-well plate with Huh7 and HepG2
  • plate 24 well plate with Hela cells for haat Elisa

19/10/2010

  • transfect miMeasure into Huh7 and HepG2 on 96-well plate again with binding sites of miR122:
    • perfect binding site
    • imperfect binding sites (randomized 9-12)
    • imperfect binding sites (randomized 9-22)
  • transfect haat cDNA constructs into Hela cells on 24-well plates
  • measurement M12-M19
    • TECAN
    • did not work, because cells were clustered
    • Confocal
    • cells from the 96 well plate were pooled together and single pictures were made.

File:M12-M22 confocalS.pdf

  • who deleted what I wrote here??? hallooo?? hmm I guess I just didn't save it. damn.

23/10/2010

  • measurement of Hela and Hek Cells transfected with miMeasure M12-M22. Four conditions were transfected with four replicates each: a = not cotransfected, b = cotransfected with miRsAg on pcDNA5, c = cotransfected with empty pcDNA5 (CMV promoter), d = cotransfected with pcDNA5 containing different shRNA (shRNA3 from Elena).
    • because of a contamination, not all replicates could be considered. Plates were screened for positive transfections
    • cells were trypsinated (30µl Trypsin) for 10min and then resuspended with 170µl PBS/BSA. Replicates for each condition were pooled into 24 well plates.
    • 200µl of each was transferred to a 96 well plate for FACS measurement.
    • 100µl of each was used for confocal analysis



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