Team:Heidelberg/Notebook/Measurement Standard

From 2010.igem.org

(Difference between revisions)
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'''[[Igem2010/Main/Measurements/Notebook/September|⇓ September]]'''
'''[[Igem2010/Main/Measurements/Notebook/September|⇓ September]]'''
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 +
'''[[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]]
 +
{{:Team:Heidelberg/Pagemiddle}}
{{:Team:Heidelberg/Pagemiddle}}
{{:Team:Heidelberg/Bottom}}
{{:Team:Heidelberg/Bottom}}

Revision as of 01:56, 24 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

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