Team:Heidelberg/Notebook/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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|- 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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<span style="color:#ffffff">-</span>
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=miMeasure=
 
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===Seeding and transfection of cells for TECAN, flow cytometry, plate reader and microscopy===
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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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5000 Hela and HEK293T 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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{| 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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Condition b: costransfection with synthetic RNA miRsAg
 
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Condition c: cotransfection with empty pcDNA5
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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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Condition d: cotransfection with synthetic shRNA3
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{{:Team:Heidelberg/Side_Bottom}}
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__NOTOC__
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A control consisting of the empty miMeasure plasmid (without binding site) was also cotransfected wit the same conditions a, b, c and d.
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=miMeasure=
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The cells were used for measurements on day three.
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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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===TECAN measurement===
 
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The
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[[Image:MiMeasure.png|500px|left|miMeasure plasmid]]
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{{:Team:Heidelberg/Bottom}}
 
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{{:Team:Heidelberg/Pagemiddle}}
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{{:Team:Heidelberg/Single_Bottom}}

Latest revision as of 03:53, 28 October 2010

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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"