Team:Heidelberg/Notebook promoters cells
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__NOTOC__ | __NOTOC__ | ||
+ | =Cell Culture Notebook= | ||
+ | =='''Contents'''== | ||
+ | |||
+ | {| class="wikitable centered" border="2" rules="rows" width="900px" style="border-color:white;" | ||
+ | |- | ||
+ | ! Week !! colspan="7" |Days | ||
+ | |- | ||
+ | |style="text-align:center"| 31 | ||
+ | |style="text-align:center"| [[Team:Heidelberg/Notebook_promoters_cells#7-27-2009|7-27-2009]] | ||
+ | |style="text-align:center"| [[Team:Heidelberg/Notebook_promoters_cells#7-28-2009|7-28-2009]] | ||
+ | |style="text-align:center"| [[Team:Heidelberg/Notebook_promoters_cells#7-29-2009|7-29-2009]] | ||
+ | |style="text-align:center"| [[Team:Heidelberg/Notebook_promoters_cells#7-30-2009|7-30-2009]] | ||
+ | |style="text-align:center"| - | ||
+ | |style="text-align:center"| - | ||
+ | |style="text-align:center"| - | ||
+ | |- | ||
+ | |style="text-align:center"| 32 | ||
+ | |style="text-align:center"| [[Team:Heidelberg/Notebook_promoters_cells#8-03-2009|8-03-2009]] | ||
+ | |style="text-align:center"| [[Team:Heidelberg/Notebook_promoters_cells#8-04-2009|8-04-2009]] | ||
+ | |style="text-align:center"| [[Team:Heidelberg/Notebook_promoters_cells#8-05-2009|8-05-2009]] | ||
+ | |style="text-align:center"| [[Team:Heidelberg/Notebook_promoters_cells#8-06-2009|8-06-2009]] | ||
+ | |style="text-align:center"| - | ||
+ | |style="text-align:center"| - | ||
+ | |style="text-align:center"| - | ||
+ | |- | ||
+ | |style="text-align:center"| 33 | ||
+ | |style="text-align:center"| - | ||
+ | |style="text-align:center"| - | ||
+ | |style="text-align:center"| - | ||
+ | |style="text-align:center"| [[Team:Heidelberg/Notebook_promoters_cells#8-13-2009|8-13-2009]] | ||
+ | |style="text-align:center"| [[Team:Heidelberg/Notebook_promoters_cells#8-14-2009|8-14-2009]] | ||
+ | |style="text-align:center"| [[Team:Heidelberg/Notebook_promoters_cells#8-15-2009|8-15-2009]] | ||
+ | |style="text-align:center"| - | ||
+ | |- | ||
+ | |style="text-align:center"| 34 | ||
+ | |style="text-align:center"| - | ||
+ | |style="text-align:center"| - | ||
+ | |style="text-align:center"| [[Team:Heidelberg/Notebook_promoters_cells#8-19-2009|8-19-2009]] | ||
+ | |style="text-align:center"| [[Team:Heidelberg/Notebook_promoters_cells#8-20-2009|8-20-2009]] | ||
+ | |style="text-align:center"| [[Team:Heidelberg/Notebook_promoters_cells#8-21-2009|8-21-2009]] | ||
+ | |style="text-align:center"| - | ||
+ | |style="text-align:center"| - | ||
+ | | | ||
+ | |- | ||
+ | |style="text-align:center"| 35 | ||
+ | |style="text-align:center"| - | ||
+ | |style="text-align:center"| - | ||
+ | |style="text-align:center"| [[Team:Heidelberg/Notebook_promoters_cells#8-26-2009|8-26-2009]] | ||
+ | |style="text-align:center"| [[Team:Heidelberg/Notebook_promoters_cells#8-27-2009|8-27-2009]] | ||
+ | |style="text-align:center"| [[Team:Heidelberg/Notebook_promoters_cells#8-28-2009|8-28-2009]] | ||
+ | |style="text-align:center"| - | ||
+ | |style="text-align:center"| [[Team:Heidelberg/Notebook_promoters_cells#8-30-2009|8-30-2009]] | ||
+ | |- | ||
+ | |style="text-align:center"| 36 | ||
+ | |style="text-align:center"| [[Team:Heidelberg/Notebook_promoters_cells#8-31-2009|8-31-2009]] | ||
+ | |style="text-align:center"| [[Team:Heidelberg/Notebook_promoters_cells#9-01-2009|9-01-2009]] | ||
+ | |style="text-align:center"| [[Team:Heidelberg/Notebook_promoters_cells#9-02-2009|9-02-2009]] | ||
+ | |style="text-align:center"| [[Team:Heidelberg/Notebook_promoters_cells#9-03-2009|9-03-2009]] | ||
+ | |style="text-align:center"| [[Team:Heidelberg/Notebook_promoters_cells#9-04-2009|9-04-2009]] | ||
+ | |style="text-align:center"| - | ||
+ | |style="text-align:center"| - | ||
+ | |- | ||
+ | |style="text-align:center"| 37 | ||
+ | |style="text-align:center"| - | ||
+ | |style="text-align:center"| - | ||
+ | |style="text-align:center"| [[Team:Heidelberg/Notebook_promoters_cells#9-09-2009|9-09-2009]] | ||
+ | |style="text-align:center"| [[Team:Heidelberg/Notebook_promoters_cells#9-10-2009|9-10-2009]] | ||
+ | |style="text-align:center"| [[Team:Heidelberg/Notebook_promoters_cells#9-11-2009|9-11-2009]] | ||
+ | |style="text-align:center"| [[Team:Heidelberg/Notebook_promoters_cells#9-12-2009|9-12-2009]] | ||
+ | |style="text-align:center"| [[Team:Heidelberg/Notebook_promoters_cells#9-13-2009|9-13-2009]] | ||
+ | |- | ||
+ | |style="text-align:center"| 38 | ||
+ | |style="text-align:center"| [[Team:Heidelberg/Notebook_promoters_cells#9-14-2009|9-14-2009]] | ||
+ | |style="text-align:center"| [[Team:Heidelberg/Notebook_promoters_cells#9-15-2009|9-15-2009]] | ||
+ | |style="text-align:center"| - | ||
+ | |style="text-align:center"| [[Team:Heidelberg/Notebook_promoters_cells#9-17-2009|9-17-2009]] | ||
+ | |style="text-align:center"| - | ||
+ | |style="text-align:center"| - | ||
+ | |style="text-align:center"| [[Team:Heidelberg/Notebook_promoters_cells#9-20-2009|9-20-2009]] | ||
+ | |- | ||
+ | |style="text-align:center"| 39 | ||
+ | |style="text-align:center"| [[Team:Heidelberg/Notebook_promoters_cells#9-21-2009|9-21-2009]] | ||
+ | |style="text-align:center"| [[Team:Heidelberg/Notebook_promoters_cells#9-22-2009|9-22-2009]] | ||
+ | |style="text-align:center"| [[Team:Heidelberg/Notebook_promoters_cells#9-23-2009|9-23-2009]] | ||
+ | |style="text-align:center"| [[Team:Heidelberg/Notebook_promoters_cells#9-24-2009|9-24-2009]] | ||
+ | |style="text-align:center"| [[Team:Heidelberg/Notebook_promoters_cells#9-25-2009|9-25-2009]] | ||
+ | |style="text-align:center"| [[Team:Heidelberg/Notebook_promoters_cells#9-26-2009|9-26-2009]] | ||
+ | |style="text-align:center"| [[Team:Heidelberg/Notebook_promoters_cells#9-27-2009|9-27-2009]] | ||
+ | |- | ||
+ | |style="text-align:center"| 40 | ||
+ | |style="text-align:center"| [[Team:Heidelberg/Notebook_promoters_cells#9-28-2009|9-28-2009]] | ||
+ | |style="text-align:center"| [[Team:Heidelberg/Notebook_promoters_cells#9-29-2009|9-29-2009]] | ||
+ | |style="text-align:center"| [[Team:Heidelberg/Notebook_promoters_cells#9-30-2009|9-30-2009]] | ||
+ | |style="text-align:center"| [[Team:Heidelberg/Notebook_promoters_cells#10-01-2009|10-01-2009]] | ||
+ | |style="text-align:center"| [[Team:Heidelberg/Notebook_promoters_cells#10-02-2009|10-02-2009]] | ||
+ | |style="text-align:center"| [[Team:Heidelberg/Notebook_promoters_cells#10-03-2009|10-03-2009]] | ||
+ | |style="text-align:center"| [[Team:Heidelberg/Notebook_promoters_cells#10-04-2009|10-04-2009]] | ||
+ | |- | ||
+ | |style="text-align:center"| 41 | ||
+ | |style="text-align:center"| [[Team:Heidelberg/Notebook_promoters_cells#10-05-2009|10-05-2009]] | ||
+ | |style="text-align:center"| [[Team:Heidelberg/Notebook_promoters_cells#10-06-2009|10-06-2009]] | ||
+ | |style="text-align:center"| [[Team:Heidelberg/Notebook_promoters_cells#10-07-2009|10-07-2009]] | ||
+ | |style="text-align:center"| [[Team:Heidelberg/Notebook_promoters_cells#10-08-2009|10-08-2009]] | ||
+ | |style="text-align:center"| [[Team:Heidelberg/Notebook_promoters_cells#10-09-2009|10-09-2009]] | ||
+ | |style="text-align:center"| [[Team:Heidelberg/Notebook_promoters_cells#10-10-2009|10-10-2009]] | ||
+ | |style="text-align:center"| [[Team:Heidelberg/Notebook_promoters_cells#10-11-2009|10-11-2009]] | ||
+ | |- | ||
+ | |style="text-align:center"| 42 | ||
+ | |style="text-align:center"| [[Team:Heidelberg/Notebook_promoters_cells#10-12-2009|10-12-2009]] | ||
+ | |style="text-align:center"| [[Team:Heidelberg/Notebook_promoters_cells#10-13-2009|10-13-2009]] | ||
+ | |style="text-align:center"| [[Team:Heidelberg/Notebook_promoters_cells#10-14-2009|10-14-2009]] | ||
+ | |style="text-align:center"| [[Team:Heidelberg/Notebook_promoters_cells#10-15-2009|10-15-2009]] | ||
+ | |style="text-align:center"| [[Team:Heidelberg/Notebook_promoters_cells#10-16-2009|10-16-2009]] | ||
+ | |style="text-align:center"| [[Team:Heidelberg/Notebook_promoters_cells#10-17-2009|10-17-2009]] | ||
+ | |style="text-align:center"| [[Team:Heidelberg/Notebook_promoters_cells#10-18-2009|10-18-2009]] | ||
+ | |- | ||
+ | |style="text-align:center"| 43 | ||
+ | |style="text-align:center"| [[Team:Heidelberg/Notebook_promoters_cells#10-19-2009|10-19-2009]] | ||
+ | |style="text-align:center"| [[Team:Heidelberg/Notebook_promoters_cells#10-20-2009|10-20-2009]] | ||
+ | |style="text-align:center"| [[Team:Heidelberg/Notebook_promoters_cells#10-21-2009|10-21-2009]] | ||
+ | |style="text-align:center"| - | ||
+ | |style="text-align:center"| - | ||
+ | |style="text-align:center"| - | ||
+ | |style="text-align:center"| - | ||
+ | |- | ||
+ | |} | ||
== 7-27-2009 == | == 7-27-2009 == | ||
Line 49: | Line 176: | ||
* 30.7.7 = 8M3 - mcherry fragment + mcherry | * 30.7.7 = 8M3 - mcherry fragment + mcherry | ||
- | == 8- | + | == 8-03-2009 == |
* split Helas --> prepared 2x8 chambers for transient transfection (1,5x10<sup>4</sup> cells/well) | * split Helas --> prepared 2x8 chambers for transient transfection (1,5x10<sup>4</sup> cells/well) | ||
- | == 8- | + | == 8-04-2009 == |
* transfected Hela in 8 chamber lab tek (morning; 1-8) with P.32 (CFP_emp_cytosole), P.34 (YFP_GPI_PM), P.31 (pcDNA5/FRT/GFP JeT), P.30 (pcDNA5/FRT/GFP Min), XXX (label washed off in centrifuge before - probably plasmid with mCherry and Min or Jet), P.6 (negative control; without fluorescent protein), mEGFPn1 (positive control) | * transfected Hela in 8 chamber lab tek (morning; 1-8) with P.32 (CFP_emp_cytosole), P.34 (YFP_GPI_PM), P.31 (pcDNA5/FRT/GFP JeT), P.30 (pcDNA5/FRT/GFP Min), XXX (label washed off in centrifuge before - probably plasmid with mCherry and Min or Jet), P.6 (negative control; without fluorescent protein), mEGFPn1 (positive control) | ||
Line 59: | Line 186: | ||
* changed medium of morning transfection (add 300 µl fresh medium) | * changed medium of morning transfection (add 300 µl fresh medium) | ||
- | == 8- | + | == 8-05-2009 == |
* changed medium of afternoon transfection from 8-4-2009 | * changed medium of afternoon transfection from 8-4-2009 | ||
* looked at all transfections --> results: targeted proteins work (but: yellow can't be distinguished from green); Min/Jet with GFP doesn't work; XXX doesn't work either | * looked at all transfections --> results: targeted proteins work (but: yellow can't be distinguished from green); Min/Jet with GFP doesn't work; XXX doesn't work either | ||
* prepared new transfections in 96 well format: 12 wells for each cell type (HeLa --> splitted 1:10 for keeping, MCF-7, U2-OS) 200µl medium with 0,75x10<sup>4</sup> cells/well | * prepared new transfections in 96 well format: 12 wells for each cell type (HeLa --> splitted 1:10 for keeping, MCF-7, U2-OS) 200µl medium with 0,75x10<sup>4</sup> cells/well | ||
- | == 8- | + | == 8-06-2009 == |
* transfection of HeLa, MCF7 and U2OS (prepared 8-5-09) with p12 (mCherry, Min promoter), p33 (YFP, cytosole), p35 (CFP, plasma membrane), p36 (YFP, ER), p37 (CFP, ER), p41 (YFP, nucleus), XXX (???, label washed off in centrifuge), p6 (negative control) and eGFP-N1 (positive control) according to effectene protocol for 96-well plate | * transfection of HeLa, MCF7 and U2OS (prepared 8-5-09) with p12 (mCherry, Min promoter), p33 (YFP, cytosole), p35 (CFP, plasma membrane), p36 (YFP, ER), p37 (CFP, ER), p41 (YFP, nucleus), XXX (???, label washed off in centrifuge), p6 (negative control) and eGFP-N1 (positive control) according to effectene protocol for 96-well plate | ||
Line 167: | Line 294: | ||
** synthetic HIF promoter (32 samples/32 controls) | ** synthetic HIF promoter (32 samples/32 controls) | ||
- | ==9- | + | ==9-01-2009== |
* removed transfection complexes and changed meidum of transfected cells | * removed transfection complexes and changed meidum of transfected cells | ||
* applied drugs to the cells: | * applied drugs to the cells: | ||
Line 194: | Line 321: | ||
* HeLa cells don't survive in minimal medium over night! -> use MCF-7 instead | * HeLa cells don't survive in minimal medium over night! -> use MCF-7 instead | ||
- | ==9- | + | ==9-02-2009== |
* prepared HeLa cells for transfection: | * prepared HeLa cells for transfection: | ||
** 24 6-wells with 10<sup>5</sup> cells/well | ** 24 6-wells with 10<sup>5</sup> cells/well | ||
Line 200: | Line 327: | ||
* 6x10<sup>6</sup> cells for RNA extraction isolated | * 6x10<sup>6</sup> cells for RNA extraction isolated | ||
- | ==9- | + | ==9-03-2009== |
* transfected HeLa according to effectene protocol: | * transfected HeLa according to effectene protocol: | ||
*transfected plasmids: | *transfected plasmids: | ||
Line 207: | Line 334: | ||
** P7_CMV_mCherry | ** P7_CMV_mCherry | ||
- | ==9- | + | ==9-09-2009== |
* transfection for p53 & NFκB screening: | * transfection for p53 & NFκB screening: | ||
** 2x28 96-wells of MCF-7 for p53 | ** 2x28 96-wells of MCF-7 for p53 | ||
Line 273: | Line 400: | ||
* made 50 µl aliquots of DFO (induces HIF --> use in combination with hypoxia bag for 24 to 48 hours) | * made 50 µl aliquots of DFO (induces HIF --> use in combination with hypoxia bag for 24 to 48 hours) | ||
- | == | + | == 9-17-2009== |
* transfections: | * transfections: | ||
** 3 24-wells of MCF-7 with p55&p43 (2:1), p31&p43 (2:1), p48&p43 (2:1) (Lipofectamine) | ** 3 24-wells of MCF-7 with p55&p43 (2:1), p31&p43 (2:1), p48&p43 (2:1) (Lipofectamine) | ||
Line 282: | Line 409: | ||
* changed medium (dye-free DMEM+++) on 6 24-wells for microscopy | * changed medium (dye-free DMEM+++) on 6 24-wells for microscopy | ||
- | == | + | == 9-20-2009== |
* changed medium of transfected cells (morning) | * changed medium of transfected cells (morning) | ||
- | == | + | == 9-21-2009== |
* prepared 12 replicates of 1,5*10<sup>5</sup> cells per promoter (Jet/CMV) | * prepared 12 replicates of 1,5*10<sup>5</sup> cells per promoter (Jet/CMV) | ||
* prepared HeLa for constitutive promoter measurement: 24 promoters, 3 controls (3 replicates for each) | * prepared HeLa for constitutive promoter measurement: 24 promoters, 3 controls (3 replicates for each) | ||
* prepared HeLa for AHR screening: 96 wells | * prepared HeLa for AHR screening: 96 wells | ||
- | == | + | == 9-22-2009== |
* prepared cells for SREBP-, PPARγ- and estrogen screening: | * prepared cells for SREBP-, PPARγ- and estrogen screening: | ||
** SREBP: 9 samples for Hela | ** SREBP: 9 samples for Hela | ||
Line 297: | Line 424: | ||
* prepared HeLa cells for constitutiv promoter meassurement: 81 96-wells | * prepared HeLa cells for constitutiv promoter meassurement: 81 96-wells | ||
- | == | + | == 9-23-2009== |
* transfection of constitutive promoters in HeLa: 3xp6, 3xp31/p6 (2:1), 3x24 constitutive promoters/p6 (2:1) | * transfection of constitutive promoters in HeLa: 3xp6, 3xp31/p6 (2:1), 3x24 constitutive promoters/p6 (2:1) | ||
* transfection of | * transfection of | ||
Line 306: | Line 433: | ||
* prepared MCF-7 for p53 transfections | * prepared MCF-7 for p53 transfections | ||
- | == | + | == 9-24-2009 == |
* transfection of MCF-7:6xp6, 6xp31/p6 (2:1), 6xp53 promoters (S2, 5, 8, 9 ,1; L1, 4, 17, 18) | * transfection of MCF-7:6xp6, 6xp31/p6 (2:1), 6xp53 promoters (S2, 5, 8, 9 ,1; L1, 4, 17, 18) | ||
* induce yesterdays transfections: | * induce yesterdays transfections: | ||
Line 316: | Line 443: | ||
* PPARγ: not induced because drug not available | * PPARγ: not induced because drug not available | ||
- | == | + | == 9-25-2009 == |
* screening | * screening | ||
** AHR at 2 pm | ** AHR at 2 pm | ||
Line 322: | Line 449: | ||
* induced p53: every two minutes 3 replicates of one promoter induced | * induced p53: every two minutes 3 replicates of one promoter induced | ||
- | == | + | == 9-26-2009 == |
*prepared cells for transfections: | *prepared cells for transfections: | ||
** Hela | ** Hela | ||
Line 329: | Line 456: | ||
** U2-OS: 24 96-wells for PPARY | ** U2-OS: 24 96-wells for PPARY | ||
- | == | + | == 9-27-2009 == |
* prepared cells for transfection SREBP, PPARγ, Estrogen and AHR again | * prepared cells for transfection SREBP, PPARγ, Estrogen and AHR again | ||
- | == | + | == 9-28-2009 == |
* prepared cells for transfections: | * prepared cells for transfections: | ||
** HeLa: 81 96-wells for constitutive promoters | ** HeLa: 81 96-wells for constitutive promoters | ||
Line 338: | Line 465: | ||
* transfected AHR/CYP, SREBP, Estrogen, PPARγ | * transfected AHR/CYP, SREBP, Estrogen, PPARγ | ||
- | == | + | == 9-29-2009 == |
* induced yesterdays transfections: | * induced yesterdays transfections: | ||
** SREBP: | ** SREBP: | ||
Line 352: | Line 479: | ||
** U2-OS: 90 96-wells for NFκB promoters | ** U2-OS: 90 96-wells for NFκB promoters | ||
- | == | + | == 9-30-2009 == |
* induced: | * induced: | ||
** put β-Estradiol on MCF-7 at 8 am -> TECAN 10-1 at 8 am (after 24 h) | ** put β-Estradiol on MCF-7 at 8 am -> TECAN 10-1 at 8 am (after 24 h) | ||
Line 364: | Line 491: | ||
** MCF-7 for HIF (FACS) | ** MCF-7 for HIF (FACS) | ||
- | == | + | == 10-01-2009 == |
* induced: | * induced: | ||
** 7 am: NFκB with TNFα (1:1000 in DMEM++/without FCS) every two minutes three replicates of one promoter -> FACS at 5 pm | ** 7 am: NFκB with TNFα (1:1000 in DMEM++/without FCS) every two minutes three replicates of one promoter -> FACS at 5 pm | ||
Line 376: | Line 503: | ||
** MCF-7 for Estrogen (TECAN) | ** MCF-7 for Estrogen (TECAN) | ||
- | == | + | == 10-02-2009 == |
* induced: | * induced: | ||
** 7 am: nfk-b with TNFα (1:1000 in DMEM++/without FCS) every two minutes three replicates of one promoter -> FACS at 5 pm | ** 7 am: nfk-b with TNFα (1:1000 in DMEM++/without FCS) every two minutes three replicates of one promoter -> FACS at 5 pm | ||
Line 390: | Line 517: | ||
** U2-OS for NFκB | ** U2-OS for NFκB | ||
- | == | + | == 10-03-2009 == |
* induced: | * induced: | ||
** PPARy for TECAN on 10-5 | ** PPARy for TECAN on 10-5 | ||
Line 402: | Line 529: | ||
** 24-wells HeLa with coverslips for microscopy | ** 24-wells HeLa with coverslips for microscopy | ||
- | == | + | == 10-04-2009 == |
* induced: | * induced: | ||
** β-Estradiol on Estrogen stuff -> TECAN 10-5 after 24 h | ** β-Estradiol on Estrogen stuff -> TECAN 10-5 after 24 h | ||
Line 412: | Line 539: | ||
** microscopy??? | ** microscopy??? | ||
- | == | + | == 10-05-2009 == |
* induced: | * induced: | ||
** 3 pm: AHR (Benzo-a-pyren) for FACS on 10-6 at 9 am | ** 3 pm: AHR (Benzo-a-pyren) for FACS on 10-6 at 9 am | ||
Line 425: | Line 552: | ||
** 2x8 96-wells U2-OS for FACS test | ** 2x8 96-wells U2-OS for FACS test | ||
- | == | + | == 10-06-2009 == |
* transfections: | * transfections: | ||
** Estrogen in MCF-7 for FACS (3 promoters) | ** Estrogen in MCF-7 for FACS (3 promoters) | ||
Line 441: | Line 568: | ||
** MCF-7 for p53 for TECAN | ** MCF-7 for p53 for TECAN | ||
- | == | + | == 10-07-2009 == |
* induced: | * induced: | ||
** NFκB in ibidi with TNFα (4.50 am; used DMEM+++ instead of DMEM++ -> TNFα probably inactivated -> "reinduced" at 8 am -> ready for fixation at 6.15 pm) | ** NFκB in ibidi with TNFα (4.50 am; used DMEM+++ instead of DMEM++ -> TNFα probably inactivated -> "reinduced" at 8 am -> ready for fixation at 6.15 pm) | ||
Line 461: | Line 588: | ||
** HeLa in 24-wells with coverslips for microscopy | ** HeLa in 24-wells with coverslips for microscopy | ||
- | == | + | == 10-08-2009 == |
* induced: | * induced: | ||
** SREBP for TECAN: changed media (full medium, inactivating, activating) at 9 am | ** SREBP for TECAN: changed media (full medium, inactivating, activating) at 9 am | ||
Line 478: | Line 605: | ||
** U2-OS for cross induction of good NFκB promoter with PPARγ drug | ** U2-OS for cross induction of good NFκB promoter with PPARγ drug | ||
- | == | + | == 10-09-2009 == |
* induce: | * induce: | ||
** natural promoters (HMG-CoA-Synthase, LDL-Rezeptor -> SREBP -> change medium, c-Jun -> put DMEM++ on cells) for TECAN | ** natural promoters (HMG-CoA-Synthase, LDL-Rezeptor -> SREBP -> change medium, c-Jun -> put DMEM++ on cells) for TECAN | ||
Line 489: | Line 616: | ||
** HeLa for constitutives and standard | ** HeLa for constitutives and standard | ||
- | == | + | == 10-10-2009 == |
* induce: | * induce: | ||
** SREBP for TECAN: HPCD at 9.30 am, remove at 12.30 pm -> TECAN at 3.30 pm | ** SREBP for TECAN: HPCD at 9.30 am, remove at 12.30 pm -> TECAN at 3.30 pm | ||
Line 505: | Line 632: | ||
** ibidi (8 wells) with 1,5x10^4 cells/well for time lapse fluorescence movie of NFκB induction | ** ibidi (8 wells) with 1,5x10^4 cells/well for time lapse fluorescence movie of NFκB induction | ||
- | == | + | == 10-11-2009 == |
* induce: | * induce: | ||
** EGF on c-Jun at 8 am -> FACS at 1 pm (one plate with CYP -> induction yesterday) | ** EGF on c-Jun at 8 am -> FACS at 1 pm (one plate with CYP -> induction yesterday) | ||
Line 520: | Line 647: | ||
** 12 24-wells of U2-OS with coverslips for microscopy | ** 12 24-wells of U2-OS with coverslips for microscopy | ||
- | == | + | == 10-12-2009 == |
* induce: | * induce: | ||
** p53 in MCF-7 once for FACS, once for TECAN (CPT for activation, Pifithrin-α for inactivation, minimal medium as reference) at 7.25 am | ** p53 in MCF-7 once for FACS, once for TECAN (CPT for activation, Pifithrin-α for inactivation, minimal medium as reference) at 7.25 am | ||
Line 531: | Line 658: | ||
** NIIL10 in U2-OS (8*ibidi) with 4* cotransfections for each NIIL10/p57, p31/p57 (ratio 2:1) | ** NIIL10 in U2-OS (8*ibidi) with 4* cotransfections for each NIIL10/p57, p31/p57 (ratio 2:1) | ||
** p53 in MCF-7 in 96-wells: 4* triplicates of each pS9 and JeT, 4*p6; triplicates of each pS9/const AMPK, pS9/dn AMPK, p31/const AMPK, p31/dn AMPK,1* each p6/const AMPK, p6/dn AMPK | ** p53 in MCF-7 in 96-wells: 4* triplicates of each pS9 and JeT, 4*p6; triplicates of each pS9/const AMPK, pS9/dn AMPK, p31/const AMPK, p31/dn AMPK,1* each p6/const AMPK, p6/dn AMPK | ||
+ | * cells: | ||
+ | ** HeLa for measurement of different time points of CMV and JeT | ||
- | == | + | == 10-13-2009 == |
* induce: | * induce: | ||
** 6-wells with NIIL10 for time course of NFκB induction -> induction: 7.45 am, freeze cells (6 induced and 6 non-induced replicates after 1 h, 6 induced replicates after 6 h and 9,5 h, 6 induced and 6 non-induced replicates after 11,75 h) | ** 6-wells with NIIL10 for time course of NFκB induction -> induction: 7.45 am, freeze cells (6 induced and 6 non-induced replicates after 1 h, 6 induced replicates after 6 h and 9,5 h, 6 induced and 6 non-induced replicates after 11,75 h) | ||
Line 542: | Line 671: | ||
** MCF-7 (24* 96-wells)standard plate | ** MCF-7 (24* 96-wells)standard plate | ||
* transfections (morning): | * transfections (morning): | ||
- | ** | + | ** HeLa: three triplicates of CMV/p57, JeT/p57, p6 for medium depend measurement of constitutive promoters |
- | ** | + | ** HeLa: two triplicates of CMV/p57, JeT/p57, p6 for time measurement of constitutive promoters |
* changed medium of transfections triplicate of CMV/p57, JeT/p57, p6 with (afternoon) | * changed medium of transfections triplicate of CMV/p57, JeT/p57, p6 with (afternoon) | ||
- | ** DMEM+++ | + | ** DMEM+++ -> FACS 10-14 (20 h) & 10-15 (50 h) |
- | ** DMEM++ | + | ** DMEM++ -> FACS 10-14 |
- | ** DMEM++ with everolimus | + | ** DMEM++ with everolimus -> FACS 10-14 |
- | == | + | == 10-14-2009 == |
* induce: 7.30 am U2-OS (8* ibidi) NFκ-B with TNFα | * induce: 7.30 am U2-OS (8* ibidi) NFκ-B with TNFα | ||
* transfections: | * transfections: | ||
** U2-OS (18* 96-wells) with triplicates of p6, JeT/p57 and NIIL10/p57 | ** U2-OS (18* 96-wells) with triplicates of p6, JeT/p57 and NIIL10/p57 | ||
- | ** U2-OS and MCF-7 (each 24* 96-wells) each with triplicates of p6, p6/p31, p6/p48 p6/p55, p6/ p57, p57/p31, p57/p48, p57/p55 (ratio of cotransfections 1:2) | + | ** U2-OS and MCF-7 (each 24* 96-wells) each with triplicates of p6, p6/p31, p6/p48 p6/p55, p6/ p57, p57/p31, p57/p48, p57/p55 (ratio of cotransfections 1:2) -> FACS 10-15 |
* prepared cells: | * prepared cells: | ||
- | ** U2-OS (8* ibidi) for NIIL10/JeT cotransfections | + | ** U2-OS (8* ibidi) for NIIL10/JeT cotransfections |
- | == | + | == 10-15-2009 == |
- | * induced U2-OS (18* 96-wells) with triplicates of p6, JeT/p57 and NIIL10/p57 at 8 am | + | * induced U2-OS (18* 96-wells) with triplicates of p6, JeT/p57 and NIIL10/p57 at 8 am -> FACS at 6 pm |
* transfections: | * transfections: | ||
** U2-OS (8* ibidi) for NIIL10/JeT cotransfections (ratio 2:1) | ** U2-OS (8* ibidi) for NIIL10/JeT cotransfections (ratio 2:1) | ||
+ | * prepared cells for sequences ordered from Geneart: | ||
+ | ** 3x60 96-wells HeLa on black plates for a maximum of 13 HB_SREBPI responsive promoters (predicted with HEARTBEAT) | ||
+ | ** 2x60 96-wells MCF-7 on black plates for a maximum of 11 HB_VDRI responsive promoters (predicted with HEARTBEAT) | ||
+ | == 10-16-2009 == | ||
+ | * cloning did not work for all the sequences | ||
+ | * transfected: | ||
+ | ** HB_SREBPI (9 wells each) in HeLa: p6, p31/p57, clones 2, 3, 4, 7, 8, 9 and 11 (each cotransfected with p57 at a 1:2 ratio) | ||
+ | ** HB_VDRI (6 wells each) in MCF-7: p6, p31/p57, clones 15?, 16, 18, 20, 21, 22, 23, 24, 25 (each cotransfected with p57 at a 1:2 ratio) | ||
+ | == 10-17-2009 == | ||
+ | * induction: | ||
+ | ** HB_SREBPI in HeLa: full medium (DMEM+++), inactivating medium (DMEM+++ +LDL +Cholesterol), activating medium (DMEM++ +LDS instead of FCS) -> HPCD and FACS: 10-20 | ||
+ | ** HB_VDRI in MCF-7: 10<sup>-7</sup>M Vitamine D3 -> TECAN every 24 h; FACS 10-21 | ||
+ | |||
+ | == 10-18-2009 == | ||
+ | * cells: | ||
+ | ** 2x60 96-wells MCF-7 for HB_VDRII | ||
+ | |||
+ | == 10-19-2009 == | ||
+ | * cells: | ||
+ | ** 2x60 96-wells HeLa for HB_SREBPII | ||
+ | ** 1 24-well of HeLa with coverslip to test if transfection also works in submission plasmid (pSB1A3, BioBricked CMV, mCherry, PM localization signal -> p53_mCherry_PM) | ||
+ | * transfections: | ||
+ | ** HB_VDRII in MCF-7: 6 wells of clones 16, 18, 20, 21, 22, 23, 24, 25; 12 wells of clones 15, 17, 19, p6, p31/p57 (all clones cotransfected as usual) | ||
+ | |||
+ | == 10-20-2009 == | ||
+ | * induce: | ||
+ | ** HB_VDRII in MCF-7: those clones with 12 replicates and controls -> 10<sup>-7</sup>M and 10<sup>-9</sup>M Vitamine D3, others (6 replicates) -> 10<sup>-9</sup>M Vitamine D3 | ||
+ | * cells: | ||
+ | ** 2x60 wells HeLa for HB_SREBPIII | ||
+ | ** 2x60 wells MCF-7 for HB_VDRIII | ||
+ | * transfections: | ||
+ | ** HB_SREBPII in HeLa: 9 wells of clones 1, 2, 3, 4, 7, 8, 9, 11 and 13; 18 wells of p6 and p31/57 (one set of controls for each plate) | ||
+ | ** 24-well (HeLa) with p53_mCherry_PM (Lipofectamine) | ||
+ | |||
+ | == 10-21-2009 == | ||
+ | * induce: | ||
+ | ** HB_SREBPII in HeLa: full medium (DMEM+++), inactivating medium (DMEM+++ +LDL +Cholesterol), activating medium (DMEM++ +LDS instead of FCS) -> HPCD and FACS: 10-24 | ||
+ | * transfections: | ||
+ | ** HB_SREBPIII: 9 wells of clones 1, 2, 3, 4, 7, 8, 9, 11, 13; 18 wells of p6 and p31/57 (one set of controls for each plate) | ||
+ | ** HB_VDRIII: 9 wells (for two concentrations: 10<sup>-7</sup>M and 10<sup>-9</sup>M of Vitamine D3) of clones 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 | ||
|} | |} |
Latest revision as of 00:29, 22 October 2009
Cell Culture NotebookContents
7-27-2009
7-28-2009
7-29-2009
7-30-2009
IDs created
8-03-2009
8-04-2009
8-05-2009
8-06-2009
8-13-2009
8-14-2009
8-15-2009
8-19-2009
8-20-2009
8-21-2009
8-26-2009
8-27-2009
8-28-2009
8-30-2009
8-31-2009
9-01-2009
9-02-2009
9-03-2009
9-09-2009
9-10-2009
==> SCREENING of p53 & NFκB 9-11-2009
9-12-2009
9-13-2009
9-14-2009
==> SCREENING --> HIF induction does not seem to work; also barely any difference between negative control and inhibited (Everolimus) samples
9-15-2009
9-16-2009
==> SCREENING (microscopy and TECAN --> approx. 8 promising promoters)
9-17-2009
9-20-2009
9-21-2009
9-22-2009
9-23-2009
9-24-2009
9-25-2009
9-26-2009
9-27-2009
9-28-2009
9-29-2009
9-30-2009
10-01-2009
10-02-2009
10-03-2009
10-04-2009
10-05-2009
10-06-2009
10-07-2009
10-08-2009
10-09-2009
10-10-2009
10-11-2009
10-12-2009
10-13-2009
10-14-2009
10-15-2009
10-16-2009
10-17-2009
10-18-2009
10-19-2009
10-20-2009
10-21-2009
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