Team:Heidelberg/Notebook/Measurement Standard
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+ | |||
+ | =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 | ||
+ | |||
Revision as of 02:02, 24 October 2010
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