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| Line 3: |
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| | =Measurement Standard= | | =Measurement Standard= |
| - | '''[[Igem2010/Main|⇐ Main Page]]'''
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| - |
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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
| |
| - | *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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| - |
| |
| - | ----
| |
| - |
| |
| - | =09/08/2010=
| |
| - |
| |
| - | seeding and transfecting cells for microscopy test measurements
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| - |
| |
| - | * add 0.6ul FuGENE reagent to 20ul of OptiMEM
| |
| - | * 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)
| |
| - | * transfere cells to the plate
| |
| - | * 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;"
| |
| - | | 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)
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| - | * leave for 30' in the incubator
| |
| - | * 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
| |
| - |
| |
| - | ----
| |
| - |
| |
| - | =25/08/2010=
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| - |
| |
| - | seeding cells for pilot FACS and Tecan measurements (96-well plate)
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| - |
| |
| - | ----
| |
| - |
| |
| - | =26/08/2010=
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| - |
| |
| - | transfection - 96-well plate
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| - |
| |
| - | ----
| |
| - |
| |
| - | =27/08/2010=
| |
| - |
| |
| - | pilot FACS and Tecan measurements (96-well plate)
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| - |
| |
| - |
| |
| - | '''[[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|⇐ Main Page]]'''
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| - |
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| - |
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| - | {| cellspacing="10" style="color:#c0c0c0"
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| - | |-
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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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| - | |-
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| - | | colspan="7"| <span style="color:#ffffff">-</span>
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| - | |}
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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/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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| - | |-
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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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| - | |}
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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/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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| - | |-
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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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| - | |'''[[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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| - | |-
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| - | |'''13'''||'''14'''||'''15'''||'''16'''||'''17'''||'''18'''||'''19'''
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| - | |-
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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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| - | |-
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| - | |'''27'''||'''28'''||'''29'''||'''30'''||colspan="3"|
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| - | |-
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| - | | colspan="7"| <span style="color:#ffffff">-</span>
| |
| - | |}
| |
| - | |
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| - | {| 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'''
| |
| - | |-
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| - | |'''4'''||'''5'''||'''6'''||'''7'''||'''8'''||'''9'''||'''10'''
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| - | |-
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| - | |'''11'''||'''12'''||'''13'''||'''14'''||'''15'''||'''16'''||'''17'''
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| - | |-
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| - | |'''18'''||'''19'''||'''20'''||'''21'''||'''22'''||'''23'''||'''24'''
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| - | |-
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| - | |'''25'''||'''26'''||'''27'''||'''28'''||'''29'''||'''30'''||'''31'''
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| - | |-
| |
| - | | colspan="7"| <span style="color:#ffffff">-</span>
| |
| - | |}
| |
| - | |}
| |
| - |
| |
| | {{:Team:Heidelberg/Bottom}} | | {{:Team:Heidelberg/Bottom}} |
"
