Team:Heidelberg/Notebook/miMeasure

From 2010.igem.org

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=miMeasure=
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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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===Seeding and transfection of cells for microscopy and flow cytometry===
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5000 Hela cells were seeded on day one in each well of the 96 well plate. Transfection of the constructs (M12-M22) with four different conditions were carried out on day two. The ratio of transfection is 1 (M construct) : 5 (stuffer/ miRsAg/ pcDNA5/ shRNA3) with a total amount of 50ng DNA.
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Condition a: cotransfection with stuffer (salmon sperm DNA)
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Condition b: cotransfection with synthetic RNA miRsAg
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Condition c: cotransfection with empty pcDNA5
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Condition d: cotransfection with synthetic shRNA3
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A control consisting of the empty miMeasure plasmid (without binding site) was also cotransfected with the same conditions a, b, c and d.
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The cells were used for measurements on day three.
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[[Image:M12-M22_HeLa_daten.jpg|thumb|400px|center|'''HeLa cells two days after transfection with miMeasure''' (A) fluorescence signal GFP channel, 8bit; (B) fluorescence signal BFP channel, 8bit; (C) merge of channels A and B, RGB (D) cells after segmentation and automated cell counting and annotation]]
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<center>
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{| class="wikitable sortable" border="0" style="text-align: left"
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|-bgcolor=#cccccc
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|+ align="top, left"|'''table 3''': Used NEB restriction enzymes.
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|sequence||binding site feature||Name/number||
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|-
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|AATTCctcagt ttactag tgc catttgttcCTGCA ||perfect binding site against miRsAg||M12
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|-
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|AATTCctcagt ttactag  acg  catttgttcCTGCA||miMeasure with randomised nucleotides 10-12||M13
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|-
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|AATTCctcagt  ttactag  taa  catttgttcCTGCA ||miMeasure with randomised nucleotides 10-12||M14
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|-
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|AATTCctcagt  ttactag  acgg  atttgttcCTGCA||miMeasure with randomised nucleotides 9-12||M15
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|-
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|AATTCctcagt  ttactag  atgt  atttgttcCTGCA||miMeasure with randomised nucleotides 9-12||M16
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|-
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|AATTCctcagt ttactagtg  g  catttgttcCTGCA ||miMeasure with mutated nucleotide 10||M17
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|-
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|AATTCctcagt ttactagtg  a  catttgttcCTGCA||miMeasure with mutated nucleotide 10||M18
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|-
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|AATTCctcagt ttactagt  a  ccatttgttcCTGCA||miMeasure with mutated nucleotide 9||M20
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|-
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|AATTCctcagt t  atg  tagtgccatttgttcCTGCA||miMeasure with mutated nucleotide 9||M22
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|-
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|-||miMeasure without any binding site||NC (negative control)
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{{:Team:Heidelberg/Pagemiddle}}
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{| cellpadding="5" cellspacing="0" style="text-align: center; color:#4e93a4; border: 3px solid #4e93a4;"
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|- border="0"
|- border="0"
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! colspan="7" style="background:#4e93a4;" | [https://2010.igem.org/Team:Heidelberg/Notebook/miMeasure/July<font color="white">July</font>]
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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:#4e93a4; color:white"
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|- style="background:#c85000; color:white"
|width="20pt"|'''M'''||width="20pt"|'''T'''||width="20pt"|'''W'''||width="20pt"|'''T'''||width="20pt"|'''F'''||width="20pt"|'''S'''||width="20pt"|'''S'''
|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"
|colspan="3"| ||'''1'''||'''2'''||'''3'''||'''4'''
|colspan="3"| ||'''1'''||'''2'''||'''3'''||'''4'''
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|- style="background:#f2f2f2; color:#78b41e"
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|'''5'''||'''6'''||'''7'''||'''8'''||'''9'''||'''10'''||'''11'''
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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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|'''12'''||'''13'''||'''14'''||'''15'''||'''16'''||'''17'''||'''18'''
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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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|-
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|- style="background:#f2f2f2; color:#78b41e"
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|'''19'''||'''20'''||'''21'''||'''22'''||'''23'''||'''24'''||'''25'''
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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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|-
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|- style="background:#f2f2f2; color:#78b41e"
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|'''26'''||'''27'''||'''28'''||'''29'''||'''30'''||'''31'''||colspan="1"|
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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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|- style="background:#f2f2f2; color:#78b41e"
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|colspan="7"|
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<span style="color:#ffffff">-</span>
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{| cellpadding="5" cellspacing="0" style="text-align: center; color:#4e93a4; border: 3px solid #4e93a4;"
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{| cellpadding="5" cellspacing="0" align="center" style="text-align: center; color:#009be1; border: 1.5px solid #000000;"
|- border="0"
|- border="0"
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! colspan="7" style="background:#4e93a4;" | [https://2010.igem.org/Team:Heidelberg/Notebook/miMeasure/August<font color="white">August</font>]
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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:#4e93a4; color:white"
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|- style="background:#f09600; color:white"
|width="20pt"|'''M'''||width="20pt"|'''T'''||width="20pt"|'''W'''||width="20pt"|'''T'''||width="20pt"|'''F'''||width="20pt"|'''S'''||width="20pt"|'''S'''
|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"
|colspan="6"| ||'''1'''
|colspan="6"| ||'''1'''
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|- style="background:#f2f2f2; color:#009be1"
|'''2'''||'''3'''||'''4'''||'''5'''||'''6'''||'''7'''||'''8'''
|'''2'''||'''3'''||'''4'''||'''5'''||'''6'''||'''7'''||'''8'''
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|- style="background:#f2f2f2; color:#009be1"
|'''9'''||'''10'''||'''11'''||'''12'''||'''13'''||'''14'''||'''15'''
|'''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'''||'''21'''||'''22'''
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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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|'''23'''||'''24'''||'''25'''||'''26'''||'''27'''||'''28'''||'''29'''
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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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|'''30'''||'''31'''||colspan="5"|
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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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{| cellpadding="5" cellspacing="0" style="text-align: center; color:#4e93a4; border: 3px solid #4e93a4;"
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{| cellpadding="5" cellspacing="0" align="center" style="text-align: center; color:#78b41e; border: 1.5px solid #333333;"
|- border="0"
|- border="0"
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! colspan="7" style="background:#4e93a4;" | [https://2010.igem.org/Team:Heidelberg/Notebook/miMeasure/September<font color="white">September</font>]
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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:#4e93a4; color:white"
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|- style="background:#009be1; color:white"
|width="20pt"|'''M'''||width="20pt"|'''T'''||width="20pt"|'''W'''||width="20pt"|'''T'''||width="20pt"|'''F'''||width="20pt"|'''S'''||width="20pt"|'''S'''
|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"| ||'''1'''||'''2'''||'''3'''||'''4'''||'''5'''
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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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|'''6'''||'''7'''||'''8'''||'''9'''||'''10'''||'''11'''||'''12'''
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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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|-
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|- style="background:#f2f2f2; color:#78b41e"
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|'''13'''||'''14'''||'''15'''||'''16'''||'''17'''||'''18'''||'''19'''
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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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|-
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|- style="background:#f2f2f2; color:#78b41e"
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|'''20'''||'''21'''||'''22'''||'''23'''||'''24'''||'''25'''||'''26'''
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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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|-
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|- style="background:#f2f2f2; color:#78b41e"
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|'''27'''||'''28'''||'''29'''||'''30'''
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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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| colspan="7"| <span style="color:#ffffff">-</span>
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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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{| cellpadding="5" cellspacing="0" style="text-align: center; color:#4e93a4; border: 3px solid #4e93a4;"
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{| cellpadding="5" cellspacing="0" align="center" style="text-align: center; color:#f09600; border: 1.5px solid #333333;"
|- border="0"
|- border="0"
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! colspan="7" style="background:#4e93a4;" | [https://2010.igem.org/Team:Heidelberg/Notebook/miMeasure/October<font color="white">October</font>]
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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:#4e93a4; color:white"
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|- style="background:#78b41e; color:white"
|width="20pt"|'''M'''||width="20pt"|'''T'''||width="20pt"|'''W'''||width="20pt"|'''T'''||width="20pt"|'''F'''||width="20pt"|'''S'''||width="20pt"|'''S'''
|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"| ||'''1'''||'''2'''||'''3'''
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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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|-
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|- style="background:#f2f2f2; color:#78b41e"
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|'''4'''||'''5'''||'''6'''||'''7'''||'''8'''||'''9'''||'''10'''
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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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|-
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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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|'''11'''||'''12'''||'''13'''||'''14'''||'''15'''||'''16'''||'''17'''
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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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|'''18'''||'''19'''||'''20'''||'''21'''||'''22'''||'''23'''||'''24'''
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|- style="background:#f2f2f2; color:#78b41e"
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|-
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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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|'''25'''||'''26'''||'''27'''||'''28'''||'''29'''||'''30'''||'''31'''
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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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{{:Team:Heidelberg/Bottom}}
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{{:Team:Heidelberg/Side_Bottom}}
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__NOTOC__
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=miMeasure=
 +
 
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miMeasure allows to quantitatively detect endogenous miRNA in different cells. To detect miRNA one should clone binding site for it into 3' UTR of GFP gene. To ensure that transfection efficiency or amount of cells do not affect result we decided to include reference fluorescence protein, BFP to allow normalisation.
 +
 
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[[Image:MiMeasure.png|500px|left|miMeasure plasmid]]
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{{:Team:Heidelberg/Single_Bottom}}

Latest revision as of 03:53, 28 October 2010

July
MTWTFSS
1234
567891011
12131415161718
19202122232425
26272829301

-


August
MTWTFSS
1
2345678
9101112131415
16171819202122
23242526272829
3031


September
MTWTFSS
12345
6789101112
13141516171819
20212223242526
2728293031

-


October
MTWTFSS
123
45 678910
11131415161718
19202122232425
2627282930

-

 

miMeasure

miMeasure allows to quantitatively detect endogenous miRNA in different cells. To detect miRNA one should clone binding site for it into 3' UTR of GFP gene. To ensure that transfection efficiency or amount of cells do not affect result we decided to include reference fluorescence protein, BFP to allow normalisation.


miMeasure plasmid

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