Team:Heidelberg/Notebook/miMeasure/August

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(New page: ===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 trypsi...)
 
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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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{| cellpadding="5" cellspacing="0" align="center" style="text-align: center; color:#4e93a4; border:1.53px solid #333333;"
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|- border="0"
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! colspan="7" style="background:#c85000;" | [https://2010.igem.org/Team:Heidelberg/Notebook/miMeasure/July<font color="white">July</font>]
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|- style="background:#c85000; 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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|- style="background:#f2f2f2; color:#78b41e"
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|colspan="3"| ||'''1'''||'''2'''||'''3'''||'''4'''
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|- style="background:#f2f2f2; color:#78b41e"
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|'''[https://2010.igem.org/Team:Heidelberg/Notebook/miMeasure/July#05.2F07.2F2010  5]'''||'''6'''||'''7'''||'''8'''||'''9'''||'''10'''||'''11'''
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|- style="background:#f2f2f2; color:#78b41e"
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|'''[https://2010.igem.org/Team:Heidelberg/Notebook/miMeasure/July#12.2F07.2F2010  12]'''||'''13'''||'''14'''||'''15'''||'''16'''||'''17'''||'''18'''
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|- style="background:#f2f2f2; color:#78b41e"
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|'''19'''||'''20'''||'''21'''||'''[https://2010.igem.org/Team:Heidelberg/Notebook/miMeasure/July#22.2F07.2F2010  22]'''||'''[https://2010.igem.org/Team:Heidelberg/Notebook/miMeasure/July#23.2F07.2F2010 23]'''||'''24'''||'''25'''
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|- style="background:#f2f2f2; color:#78b41e"
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|'''[https://2010.igem.org/Team:Heidelberg/Notebook/miMeasure/July#26.2F07.2F2010  26]'''||'''[https://2010.igem.org/Team:Heidelberg/Notebook/miMeasure/July#27.2F07.2F2010 27]'''||'''28'''||'''29'''||'''30'''||'''[https://2010.igem.org/Team:Heidelberg/Notebook/miMeasure/July#31.2F07.2F20103 1]'''||colspan="1"|
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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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{| cellpadding="5" cellspacing="0" align="center" style="text-align: center; color:#009be1; border: 1.5px solid #000000;"
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|- border="0"
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! colspan="7" style="background:#f09600;" | [https://2010.igem.org/Team:Heidelberg/Notebook/miMeasure/August<font color="white">August</font>]
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|- style="background:#f09600; 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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|- style="background:#f2f2f2; color:#009be1"
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|colspan="6"| ||'''1'''
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|- style="background:#f2f2f2; color:#009be1"
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|'''2'''||'''3'''||'''4'''||'''5'''||'''6'''||'''7'''||'''8'''
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|- style="background:#f2f2f2; color:#009be1"
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|'''9'''||'''10'''||'''11'''||'''12'''||'''13'''||'''14'''||'''15'''
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|- style="background:#f2f2f2; color:#009be1"
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|'''16'''||'''17'''||'''18'''||'''19'''||'''20'''||'''[https://2010.igem.org/Team:Heidelberg/Notebook/miMeasure/August#21/08/2010 21]'''||'''[https://2010.igem.org/Team:Heidelberg/Notebook/miMeasure/August#22.2F08.2F2010 22]'''
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|- style="background:#f2f2f2; color:#009be1"
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|'''[https://2010.igem.org/Team:Heidelberg/Notebook/miMeasure/August#23.2F08.2F2010 23]'''||'''[https://2010.igem.org/Team:Heidelberg/Notebook/miMeasure/August#24.2F08.2F2010 24]'''||'''[https://2010.igem.org/Team:Heidelberg/Notebook/miMeasure/August#25.2F08.2F2010 25]'''||'''[https://2010.igem.org/Team:Heidelberg/Notebook/miMeasure/August#26.2F08.2F2010 26]'''||'''[https://2010.igem.org/Team:Heidelberg/Notebook/miMeasure/August#27.2F08.2F2010 27]'''||'''[https://2010.igem.org/Team:Heidelberg/Notebook/miMeasure/August#28.2F08.2F2010 28]'''||'''[https://2010.igem.org/Team:Heidelberg/Notebook/miMeasure/August#29.2F08.2F2010 29]'''
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|- style="background:#f2f2f2; color:#009be1"
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|[https://2010.igem.org/Team:Heidelberg/Notebook/miMeasure/August#302F08.2F2010 30]'''||[https://2010.igem.org/Team:Heidelberg/Notebook/miMeasure/August#31.2F08.2F2010 31]'''||colspan="5"|
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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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{| cellpadding="5" cellspacing="0" align="center" style="text-align: center; color:#78b41e; border: 1.5px solid #333333;"
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|- border="0"
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! colspan="7" style="background:#009be1;" | [https://2010.igem.org/Team:Heidelberg/Notebook/miMeasure/September<font color="#ffecba">September</font>]
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|- style="background:#009be1; 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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|- style="background:#f2f2f2; color:#78b41e"
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|colspan="2"| ||'''[https://2010.igem.org/Team:Heidelberg/Notebook/miMeasure/September#01.2F09.2F2010 1]'''||'''[https://2010.igem.org/Team:Heidelberg/Notebook/miMeasure/September#02.2F09.2F2010 2]'''||'''[https://2010.igem.org/Team:Heidelberg/Notebook/miMeasure/September#03.2F09.2F2010 3]'''||'''[https://2010.igem.org/Team:Heidelberg/Notebook/miMeasure/September#04.2F09.2F2010 4]'''||'''[https://2010.igem.org/Team:Heidelberg/Notebook/miMeasure/September#05.2F09.2F2010 5]'''
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|- style="background:#f2f2f2; color:#78b41e"
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|'''[https://2010.igem.org/Team:Heidelberg/Notebook/miMeasure/September#06.2F09.2F2010 6]'''||'''[https://2010.igem.org/Team:Heidelberg/Notebook/miMeasure/September#07.2F09.2F2010 7]'''||'''[https://2010.igem.org/Team:Heidelberg/Notebook/miMeasure/September#08.2F09.2F2010 8]'''||'''[https://2010.igem.org/Team:Heidelberg/Notebook/miMeasure/September#09.2F09.2F2010 9]'''||'''[https://2010.igem.org/Team:Heidelberg/Notebook/miMeasure/September#10.2F09.2F2010 10]'''||'''[https://2010.igem.org/Team:Heidelberg/Notebook/miMeasure/September#11.2F09.2F2010 11]'''||'''[https://2010.igem.org/Team:Heidelberg/Notebook/miMeasure/September#12.2F09.2F2010 12]'''
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|- style="background:#f2f2f2; color:#78b41e"
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|'''[https://2010.igem.org/Team:Heidelberg/Notebook/miMeasure/September#13.2F09.2F2010 13]'''||'''[https://2010.igem.org/Team:Heidelberg/Notebook/miMeasure/September#14.2F09.2F2010 14]'''||'''[https://2010.igem.org/Team:Heidelberg/Notebook/miMeasure/September#15.2F09.2F2010 15]'''||'''[https://2010.igem.org/Team:Heidelberg/Notebook/miMeasure/September#16.2F09.2F2010 16]'''||'''[https://2010.igem.org/Team:Heidelberg/Notebook/miMeasure/September#17.2F09.2F2010 17]'''||'''[https://2010.igem.org/Team:Heidelberg/Notebook/miMeasure/September#18.2F09.2F2010 18]'''||'''[https://2010.igem.org/Team:Heidelberg/Notebook/miMeasure/September#19.2F09.2F2010 19]'''
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|- style="background:#f2f2f2; color:#78b41e"
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|'''[https://2010.igem.org/Team:Heidelberg/Notebook/miMeasure/September#20.2F09.2F2010 20]'''||'''[https://2010.igem.org/Team:Heidelberg/Notebook/miMeasure/September#21.2F09.2F2010 21]'''||'''[https://2010.igem.org/Team:Heidelberg/Notebook/miMeasure/September#22.2F09.2F2010 22]'''||'''[https://2010.igem.org/Team:Heidelberg/Notebook/miMeasure/September#23.2F09.2F2010 23]'''||'''[https://2010.igem.org/Team:Heidelberg/Notebook/miMeasure/September#24.2F09.2F2010 24]'''||'''[https://2010.igem.org/Team:Heidelberg/Notebook/miMeasure/September#25.2F09.2F2010 25]'''||'''[https://2010.igem.org/Team:Heidelberg/Notebook/miMeasure/September#26.2F09.2F2010 26]'''
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|- style="background:#f2f2f2; color:#78b41e"
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|'''[https://2010.igem.org/Team:Heidelberg/Notebook/miMeasure/September#27.2F09.2F2010 27]'''||'''[https://2010.igem.org/Team:Heidelberg/Notebook/miMeasure/September#28.2F09.2F2010 28]'''||'''[https://2010.igem.org/Team:Heidelberg/Notebook/miMeasure/September#29.2F09.2F2010 29]'''||'''[https://2010.igem.org/Team:Heidelberg/Notebook/miMeasure/September#30.2F30.2F2010 30]'''||'''[https://2010.igem.org/Team:Heidelberg/Notebook/Homology_Based/August#31.2F08.2F2010 31]'''||colspan="5"|
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|- style="background:#f2f2f2; color:#78b41e"
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|colspan="7"|
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<span style="color:#ffffff">-</span>
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|}
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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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{| cellpadding="5" cellspacing="0" align="center" style="text-align: center; color:#f09600; border: 1.5px solid #333333;"
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|- border="0"
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! colspan="7" style="background:#78b41e;" | [https://2010.igem.org/Team:Heidelberg/Notebook/MiMeasure/October<font color="white">October</font>]
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|- style="background:#78b41e; 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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|- style="background:#f2f2f2; color:#78b41e"
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|colspan="4"| ||'''[https://2010.igem.org/Team:Heidelberg/Notebook/MiMeasure/October#01.2F10.2F2010 1]'''||'''[https://2010.igem.org/Team:Heidelberg/Notebook/MiMeasure/October#02.2F10.2F2010 2]'''||'''[https://2010.igem.org/Team:Heidelberg/Notebook/MiMeasure/October#03.2F10.2F2010 3]'''
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|- style="background:#f2f2f2; color:#78b41e"
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|'''[https://2010.igem.org/Team:Heidelberg/Notebook/MiMeasure/October#04.2F10.2F2010 4]'''||'''[https://2010.igem.org/Team:Heidelberg/Notebook/MiMeasure/October#05.2F10.2F2010 5]'''
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|'''[https://2010.igem.org/Team:Heidelberg/Notebook/MiMeasure/October#06.2F10.2F2010 6]'''||'''[https://2010.igem.org/Team:Heidelberg/Notebook/MiMeasure/October#07.2F10.2F2010 7]'''||'''[https://2010.igem.org/Team:Heidelberg/Notebook/MiMeasure/October#08.2F10.2F2010 8]'''||'''[https://2010.igem.org/Team:Heidelberg/Notebook/MiMeasure/October#09.2F10.2F2010 9]'''||'''[https://2010.igem.org/Team:Heidelberg/Notebook/MiMeasure/October#10.2F10.2F2010 10]'''
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|- style="background:#f2f2f2; color:#78b41e"
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|'''[https://2010.igem.org/Team:Heidelberg/Notebook/MiMeasure/October#11.2F10.2F2010 11]'''||'''[https://2010.igem.org/Team:Heidelberg/Notebook/MiMeasure/October#12.2F10.2F2010 12]'''|'''[https://2010.igem.org/Team:Heidelberg/Notebook/MiMeasure/October#13.2F10.2F2010 13]'''||'''[https://2010.igem.org/Team:Heidelberg/Notebook/MiMeasure/October#14.2F10.2F2010 14]'''||'''[https://2010.igem.org/Team:Heidelberg/Notebook/MiMeasure/October#15.2F10.2F2010 15]'''||'''[https://2010.igem.org/Team:Heidelberg/Notebook/MiMeasure/October#16.2F10.2F2010 16]'''||'''[https://2010.igem.org/Team:Heidelberg/Notebook/MiMeasure/October#17.2F10.2F2010 17]'''||'''[https://2010.igem.org/Team:Heidelberg/Notebook/MiMeasure/October#18.2F10.2F2010 18]'''
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|- style="background:#f2f2f2; color:#78b41e"
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|'''[https://2010.igem.org/Team:Heidelberg/Notebook/MiMeasure/October#19.2F10.2F2010 19]'''||'''[https://2010.igem.org/Team:Heidelberg/Notebook/MiMeasure/October#20.2F10.2F2010 20]'''||'''[https://2010.igem.org/Team:Heidelberg/Notebook/MiMeasure/October#21.2F10.2F2010 21]'''||'''[https://2010.igem.org/Team:Heidelberg/Notebook/MiMeasure/October#22.2F10.2F2010 22]'''||'''[https://2010.igem.org/Team:Heidelberg/Notebook/MiMeasure/October#23.2F10.2F2010 23]'''||'''[https://2010.igem.org/Team:Heidelberg/Notebook/MiMeasure/October#24.2F10.2F2010 24]'''||'''[https://2010.igem.org/Team:Heidelberg/Notebook/MiMeasure/October#25.2F10.2F2010 25]'''
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|- style="background:#f2f2f2; color:#78b41e"
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|'''[https://2010.igem.org/Team:Heidelberg/Notebook/MiMeasure/October#26.2F10.2F2010 26]'''||'''[https://2010.igem.org/Team:Heidelberg/Notebook/MiMeasure/October#27.2F10.2F2010 27]'''||'''[https://2010.igem.org/Team:Heidelberg/Notebook/MiMeasure/October#28.2F10.2F2010 28]'''||'''[https://2010.igem.org/Team:Heidelberg/Notebook/MiMeasure/October#29.2F10.2F2010 29]'''||'''[https://2010.igem.org/Team:Heidelberg/Notebook/MiMeasure/October#22.2F30.2F2010 30]'''||colspan="3"|
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|- style="background:#f2f2f2; color:#78b41e"
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|colspan="7"|
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<span style="color:#ffffff">-</span>
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|}
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{{:Team:Heidelberg/Side_Bottom}}
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__NOTOC__
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===10/08/2010===
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===08/08/2010===
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coating plates with poly-L-lysine
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seeding cells for test measurements - 96 well plate for plate-reader and FACS
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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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test of media:
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*OptiMEM 0% FBS + Pen/Strep + L-Glu + other according to cell line
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*OptiMEM 2% FBS + Pen/Strep + L-Glu + other according to cell line
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===25/08/2010===
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*DMEM 10% FBS+ Pen/Strep + L-Glu + other according to cell line
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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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* cells did not adhere properly so we decided to coat 96-well plate with poly-L-lysine and repeat
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* significantly better growth in DMEM with 10% FBS, so we decided to grow cells in it and before measurement to change it to OptiMEM, to reduce high background fluorescence coming from FBS.
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===27/08/2010===
 
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pilot FACS and Tecan measurements (96-well plate)
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{{:Team:Heidelberg/Bottom}}

Latest revision as of 09:01, 27 October 2010

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08/08/2010

seeding cells for test measurements - 96 well plate for plate-reader and FACS

test of media:

  • OptiMEM 0% FBS + Pen/Strep + L-Glu + other according to cell line
  • OptiMEM 2% FBS + Pen/Strep + L-Glu + other according to cell line
  • DMEM 10% FBS+ Pen/Strep + L-Glu + other according to cell line


  • cells did not adhere properly so we decided to coat 96-well plate with poly-L-lysine and repeat
  • significantly better growth in DMEM with 10% FBS, so we decided to grow cells in it and before measurement to change it to OptiMEM, to reduce high background fluorescence coming from FBS.


   

Retrieved from "http://2010.igem.org/Team:Heidelberg/Notebook/miMeasure/August"