Team:Heidelberg/Notebook/Measurement Standard

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

Revision as of 02:20, 24 October 2010

Measurement Standard