Team:Heidelberg/Notebook/BSDesign/October

From 2010.igem.org

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{{:Team:Heidelberg/Single_Pagetop|note_BSDesign}}
{{:Team:Heidelberg/Single_Pagetop|note_BSDesign}}
{{:Team:Heidelberg/Side_Top}}
{{:Team:Heidelberg/Side_Top}}
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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/BSDesign/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:#f09600"
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|colspan="3"| ||'''1'''||'''2'''||'''3'''||'''4'''
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|- style="background:#f2f2f2; color:#f09600"
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|'''[https://2010.igem.org/Team:Heidelberg/Notebook/BSDesign/July#05.2F07.2F2010_-_11.2F07.2F2010 5]'''||'''[https://2010.igem.org/Team:Heidelberg/Notebook/BSDesign/July#05.2F07.2F2010_-_11.2F07.2F2010 6]'''||'''[https://2010.igem.org/Team:Heidelberg/Notebook/BSDesign/July#05.2F07.2F2010_-_11.2F07.2F2010 7]'''||'''[https://2010.igem.org/Team:Heidelberg/Notebook/BSDesign/July#05.2F07.2F2010_-_11.2F07.2F2010 8]'''||'''[https://2010.igem.org/Team:Heidelberg/Notebook/BSDesign/July#05.2F07.2F2010_-_11.2F07.2F2010 9]'''||'''[https://2010.igem.org/Team:Heidelberg/Notebook/BSDesign/July#05.2F07.2F2010_-_11.2F07.2F2010 10]'''||'''[https://2010.igem.org/Team:Heidelberg/Notebook/BSDesign/July#05.2F07.2F2010_-_11.2F07.2F2010 11]'''
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|- style="background:#f2f2f2; color:#f09600"
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|'''[https://2010.igem.org/Team:Heidelberg/Notebook/BSDesign/July#12.2F07.2F2010_-_19.2F07.2F2010 12]'''||'''[https://2010.igem.org/Team:Heidelberg/Notebook/BSDesign/July#12.2F07.2F2010_-_19.2F07.2F2010 13]'''||'''[https://2010.igem.org/Team:Heidelberg/Notebook/BSDesign/July#12.2F07.2F2010_-_19.2F07.2F2010 14]'''||'''[https://2010.igem.org/Team:Heidelberg/Notebook/BSDesign/July#12.2F07.2F2010_-_19.2F07.2F2010 15]'''||'''[https://2010.igem.org/Team:Heidelberg/Notebook/BSDesign/July#12.2F07.2F2010_-_19.2F07.2F2010 16]'''||'''[https://2010.igem.org/Team:Heidelberg/Notebook/BSDesign/July#12.2F07.2F2010_-_19.2F07.2F2010 17]'''||'''[https://2010.igem.org/Team:Heidelberg/Notebook/BSDesign/July#12.2F07.2F2010_-_19.2F07.2F2010 18]'''
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|- style="background:#f2f2f2; color:#f09600"
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|'''[https://2010.igem.org/Team:Heidelberg/Notebook/BSDesign/July#12.2F07.2F2010_-_19.2F07.2F2010 19]'''||'''[https://2010.igem.org/Team:Heidelberg/Notebook/BSDesign/July#20.2F07.2F2010 20]'''||'''[https://2010.igem.org/Team:Heidelberg/Notebook/BSDesign/July#21.2F07.2F2010 21]'''||'''[https://2010.igem.org/Team:Heidelberg/Notebook/BSDesign/July#22.2F07.2F2010 22]'''||'''[https://2010.igem.org/Team:Heidelberg/Notebook/BSDesign/July#23.2F07.2F2010 23]'''||'''24'''||'''25'''
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|- style="background:#f2f2f2; color:#f09600"
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|'''[https://2010.igem.org/Team:Heidelberg/Notebook/BSDesign/July#26.2F07.2F2010 26]'''||'''[https://2010.igem.org/Team:Heidelberg/Notebook/BSDesign/July#27.2F07.2F2010 27]'''||'''[https://2010.igem.org/Team:Heidelberg/Notebook/BSDesign/July#28.2F07.2F2010 28]'''||'''[https://2010.igem.org/Team:Heidelberg/Notebook/BSDesign/July#29.2F07.2F2010 29]'''||'''30'''||'''[https://2010.igem.org/Team:Heidelberg/Notebook/BSDesign/July#31.2F07.2F2010 31]'''||
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|- style="background:#f2f2f2; color:#f09600"
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| colspan="7"|
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<span style="color:#ffffff">-</span>
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|}
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{| cellpadding="5" cellspacing="0" align="center" style="text-align: center; color:#f09600; 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/BSDesign/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:#f09600"
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|colspan="6"| ||'''[https://2010.igem.org/Team:Heidelberg/Notebook/BSDesign/August#01.2F08.2F2010 1]'''
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|- style="background:#f2f2f2; color:#f09600"
 +
|'''[https://2010.igem.org/Team:Heidelberg/Notebook/BSDesign/August#02.2F08.2F2010 2]'''||'''[https://2010.igem.org/Team:Heidelberg/Notebook/BSDesign/August#03.2F08.2F2010 3]'''||'''[https://2010.igem.org/Team:Heidelberg/Notebook/BSDesign/August#04.2F08.2F2010 4]'''||'''[https://2010.igem.org/Team:Heidelberg/Notebook/BSDesign/August#05.2F08.2F2010 5]'''||'''[https://2010.igem.org/Team:Heidelberg/Notebook/BSDesign/August#06.2F08.2F2010 6]'''||'''[https://2010.igem.org/Team:Heidelberg/Notebook/BSDesign/August#07.2F08.2F2010 7]'''||'''[https://2010.igem.org/Team:Heidelberg/Notebook/BSDesign/August#08.2F08.2F2010 8]'''
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|- style="background:#f2f2f2; color:#f09600"
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|'''[https://2010.igem.org/Team:Heidelberg/Notebook/BSDesign/August#09.2F08.2F2010 9]'''||'''[https://2010.igem.org/Team:Heidelberg/Notebook/BSDesign/August#10.2F08.2F2010 10]'''||'''[https://2010.igem.org/Team:Heidelberg/Notebook/BSDesign/August#11.2F08.2F2010 11]'''||'''12'''||'''13'''||'''14'''||'''15'''
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|- style="background:#f2f2f2; color:#f09600"
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|'''16'''||'''17'''||'''18'''||'''19'''||'''20'''||'''21'''||'''22'''
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|- style="background:#f2f2f2; color:#f09600"
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|'''23'''||'''24'''||'''25'''||'''26'''||'''27'''||'''28'''||'''29'''
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|- style="background:#f2f2f2; color:#f09600"
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|'''30'''||'''31'''||colspan="5"|
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|}
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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:#009be1;" | [https://2010.igem.org/Team:Heidelberg/Notebook/BSDesign/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:#f09600"
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|colspan="2"| ||'''1'''||'''2'''||'''3'''||'''4'''||'''5'''
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|- style="background:#f2f2f2; color:#f09600"
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|'''6'''||'''7'''||'''[https://2010.igem.org/Team:Heidelberg/Notebook/BSDesign/September#08.2F09.2F2010 8]'''||'''[https://2010.igem.org/Team:Heidelberg/Notebook/BSDesign/September#09.2F09.2F2010 9]'''||'''[https://2010.igem.org/Team:Heidelberg/Notebook/BSDesign/September#10.2F09.2F2010 10]'''||'''[https://2010.igem.org/Team:Heidelberg/Notebook/BSDesign/September#11.2F09.2F2010 11]'''||'''[https://2010.igem.org/Team:Heidelberg/Notebook/BSDesign/September#12.2F09.2F2010 12]'''
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|- style="background:#f2f2f2; color:#f09600"
 +
|'''[https://2010.igem.org/Team:Heidelberg/Notebook/BSDesign/September#13.2F09.2F2010 13]'''||'''[https://2010.igem.org/Team:Heidelberg/Notebook/BSDesign/September#14.2F09.2F2010 14]'''||'''[https://2010.igem.org/Team:Heidelberg/Notebook/BSDesign/September#15.2F09.2F2010 15]'''||'''[https://2010.igem.org/Team:Heidelberg/Notebook/BSDesign/September#16.2F09.2F2010 16]'''||'''[https://2010.igem.org/Team:Heidelberg/Notebook/BSDesign/September#17.2F09.2F2010 17]'''||'''[https://2010.igem.org/Team:Heidelberg/Notebook/BSDesign/September#18.2F09.2F2010 18]'''||'''[https://2010.igem.org/Team:Heidelberg/Notebook/BSDesign/September#19.2F09.2F2010 19]'''
 +
|- style="background:#f2f2f2; color:#f09600"
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|'''[https://2010.igem.org/Team:Heidelberg/Notebook/BSDesign/September#20.2F09.2F2010 20]'''||'''[https://2010.igem.org/Team:Heidelberg/Notebook/BSDesign/September#21.2F09.2F2010 21]'''||'''[https://2010.igem.org/Team:Heidelberg/Notebook/BSDesign/September#22.2F09.2F2010 22]'''||'''[https://2010.igem.org/Team:Heidelberg/Notebook/BSDesign/September#23.2F09.2F2010 23]'''||'''[https://2010.igem.org/Team:Heidelberg/Notebook/BSDesign/September#24.2F09.2F2010 24]'''||'''[https://2010.igem.org/Team:Heidelberg/Notebook/BSDesign/September#25.2F09.2F2010 25]'''||'''[https://2010.igem.org/Team:Heidelberg/Notebook/BSDesign/September#26.2F09.2F2010 26]'''
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|- style="background:#f2f2f2; color:#f09600"
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|'''[https://2010.igem.org/Team:Heidelberg/Notebook/BSDesign/September#27.2F09.2F2010 27]'''||'''[https://2010.igem.org/Team:Heidelberg/Notebook/BSDesign/September#28.2F09.2F2010 28]'''||'''[https://2010.igem.org/Team:Heidelberg/Notebook/BSDesign/September#29.2F09.2F2010 29]'''||'''[https://2010.igem.org/Team:Heidelberg/Notebook/BSDesign/September#30.2F30.2F2010 30]'''||colspan="5"|
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|- style="background:#f2f2f2; color:#f09600"
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|colspan="7"|
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<span style="color:#ffffff">-</span>
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|}
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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/BSDesign/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:#f09600"
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|colspan="4"| ||'''[https://2010.igem.org/Team:Heidelberg/Notebook/BSDesign/October#01.2F10.2F2010 1]'''||'''[https://2010.igem.org/Team:Heidelberg/Notebook/BSDesign/October#02.2F10.2F2010 2]'''||'''[https://2010.igem.org/Team:Heidelberg/Notebook/BSDesign/October#03.2F10.2F2010 3]'''
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|- style="background:#f2f2f2; color:#f09600"
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|'''[https://2010.igem.org/Team:Heidelberg/Notebook/BSDesign/October#04.2F10.2F2010 4]'''||'''[https://2010.igem.org/Team:Heidelberg/Notebook/BSDesign/October#05.2F10.2F2010 5]'''||'''[https://2010.igem.org/Team:Heidelberg/Notebook/BSDesign/October#06.2F10.2F2010 6]'''||'''[https://2010.igem.org/Team:Heidelberg/Notebook/BSDesign/October#07.2F10.2F2010 7]'''||'''[https://2010.igem.org/Team:Heidelberg/Notebook/BSDesign/October#08.2F10.2F2010 8]'''||'''[https://2010.igem.org/Team:Heidelberg/Notebook/BSDesign/October#09.2F10.2F2010 9]'''||'''[https://2010.igem.org/Team:Heidelberg/Notebook/BSDesign/October#10.2F10.2F2010 10]'''
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|- style="background:#f2f2f2; color:#f09600"
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|'''[https://2010.igem.org/Team:Heidelberg/Notebook/BSDesign/October#11.2F10.2F2010 11]'''||'''[https://2010.igem.org/Team:Heidelberg/Notebook/BSDesign/October#12.2F10.2F2010 12]'''||'''[https://2010.igem.org/Team:Heidelberg/Notebook/BSDesign/October#13.2F10.2F2010 13]'''||'''[https://2010.igem.org/Team:Heidelberg/Notebook/BSDesign/October#14.2F10.2F2010 14]'''||'''[https://2010.igem.org/Team:Heidelberg/Notebook/BSDesign/October#15.2F10.2F2010 15]'''||'''[https://2010.igem.org/Team:Heidelberg/Notebook/BSDesign/October#16.2F10.2F2010 16]'''||'''[https://2010.igem.org/Team:Heidelberg/Notebook/BSDesign/October#17.2F10.2F2010 17]'''
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|- style="background:#f2f2f2; color:#f09600"
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|'''[https://2010.igem.org/Team:Heidelberg/Notebook/BSDesign/October#18.2F10.2F2010 18]'''|||'''[https://2010.igem.org/Team:Heidelberg/Notebook/BSDesign/October#19.2F10.2F2010 19]'''||'''[https://2010.igem.org/Team:Heidelberg/Notebook/BSDesign/October#20.2F10.2F2010 20]'''||'''[https://2010.igem.org/Team:Heidelberg/Notebook/BSDesign/October#21.2F10.2F2010 21]'''||'''[https://2010.igem.org/Team:Heidelberg/Notebook/BSDesign/October#22.2F10.2F2010 22]'''||'''[https://2010.igem.org/Team:Heidelberg/Notebook/BSDesign/October#23.2F10.2F2010 23]'''||'''24'''
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|- style="background:#f2f2f2; color:#f09600"
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|'''25'''||'''26'''||'''27'''||'''28'''||'''29'''||'''30'''||'''31'''
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|- style="background:#f2f2f2; color:#f09600"
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|colspan="7"|
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<span style="color:#ffffff">-</span>
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|}
{{:Team:Heidelberg/Side_Bottom}}
{{:Team:Heidelberg/Side_Bottom}}
Line 8: Line 90:
= Binding Site Design - October =
= Binding Site Design - October =
 +
== 02/10/2010 ==
 +
<br />
 +
*digestion in 50 µl of
 +
**4x pSB1C3 (5µl BSA, 5µl NEB buffer 2, 1µl PstI, 1µl EcoRI, 1µl DpnI, 4µl construct, 33 µl H2O)
 +
**4x pSB1A3 (5µl BSA, 5µl NEB buffer 2, 1µl PstI, 1µl EcoRI, 1µl DpnI, 4µl construct, 33 µl H2O)
 +
 +
== 05/10/2010 ==
 +
<br />
 +
*1µg digestion of miMeasure and 6 different constructs (1A, 3F, 13, 15, 17, 37) with EcoRI and PstI
 +
 +
:1= 0S
 +
:2= 0L
 +
:3= 10S
 +
:4= 10L
 +
 +
 +
== 06/10/2010 ==
 +
 +
20 Miniprep:
 +
{| class="wikitable sortable" border="0" align="center" style="text-align: left"
 +
|-bgcolor=#009be1
 +
|+ align="top, left"|'''Table 1: 20 Minis'''
 +
|Number||binding site against miRNA x||Position
 +
|-
 +
|1||221||3.5
 +
|-
 +
|2||221||1.4
 +
|-
 +
|3||1179||3.1
 +
|-
 +
|4||4286||2.6
 +
|-
 +
|5||4286||4.1
 +
|-
 +
|6||4286||3.4
 +
|-
 +
|7||1179||4.4
 +
|-
 +
|8||1179|3.5
 +
|-
 +
|9||1179||1.9
 +
|-
 +
|10||221||4.6
 +
|-
 +
|11||221||1.5
 +
|-
 +
|12||4286||3.5
 +
|-
 +
|13||221||4.8
 +
|-
 +
|14||221||2.3
 +
|-
 +
|15||4286||2.2
 +
|-
 +
|16||4286||1.3
 +
|-
 +
|17||1179||2.5
 +
|-
 +
|18||1179||2.4
 +
|-
 +
|19||1179||1.5
 +
|-
 +
|20||1179||4.8
 +
|}
 +
 +
* digestion of miMeasure and 6 different synthetic miRNA patterns against endogenous miR122
 +
* gel seperation
 +
* gel purification
 +
* sequencing
 +
----
 +
 +
 +
== 12/10/2010 ==
 +
 +
4 miraPCRs for binding sites of miR4286.
 +
* one PCR with an annealing temperature of 45 degree and 16 cycles
 +
* second miraPCR with annealing temperature of 57 degree and also 16 cycles
 +
**nucleotide removal
 +
**digestion
 +
**gel seperation
 +
**gel purification
 +
**ligation in miMeasure and pSB1C3
 +
**transformation
 +
 +
== 13/10/2010 ==
 +
 +
*pick colonies
 +
*about 8 hours later: do minipreps
 +
*send for sequencing
 +
{{:Team:Heidelberg/Single_Bottom}}
 +
 +
----
 +
 +
= 14/10/2010 =
 +
* a reasonable
 +
* Screening of 40 colonies of miR4286 (colony PCR)
 +
* Screening of 32 colonies of miR320b (colony PCR)
 +
 +
 +
all negative
 +
<br /><br /><br />
 +
 +
----
 +
 +
= 15/10/2010 =
 +
 +
ligation of miMeasure construct:<br /><br />
 +
 +
miR-122<br />
 +
miR-122ran<br />
 +
miR-1179<br />
 +
miR-221<br />
 +
<br />
 +
each in 0S/0L/10S/10L versions
 +
 +
<br /><br />
 +
 +
----
 +
 +
= 16/10/2010 =
 +
transformation of each ligation to TOP10 ''E.coli''
 +
 +
cells were plated on LB-amp plates
 +
<br /><br /><br />
 +
 +
----
 +
 +
= 17/10/2010 =
 +
96 colony PCRs - 8 from each plate
 +
<br>
 +
ALL NEGATIVE!!!!
 +
<br /><br /><br />
----
----
{{:Team:Heidelberg/Single_Bottom}}
{{:Team:Heidelberg/Single_Bottom}}

Latest revision as of 03:58, 28 October 2010

July
MTWTFSS
1234
567891011
12131415161718
19202122232425
262728293031

-


August
MTWTFSS
1
2345678
9101112131415
16171819202122
23242526272829
3031


September
MTWTFSS
12345
6789101112
13141516171819
20212223242526
27282930

-


October
MTWTFSS
123
45678910
11121314151617
18192021222324
25262728293031

-

 


Binding Site Design - October

02/10/2010


  • digestion in 50 µl of
    • 4x pSB1C3 (5µl BSA, 5µl NEB buffer 2, 1µl PstI, 1µl EcoRI, 1µl DpnI, 4µl construct, 33 µl H2O)
    • 4x pSB1A3 (5µl BSA, 5µl NEB buffer 2, 1µl PstI, 1µl EcoRI, 1µl DpnI, 4µl construct, 33 µl H2O)

05/10/2010


  • 1µg digestion of miMeasure and 6 different constructs (1A, 3F, 13, 15, 17, 37) with EcoRI and PstI
1= 0S
2= 0L
3= 10S
4= 10L


06/10/2010

20 Miniprep:

Table 1: 20 Minis
Numberbinding site against miRNA xPosition
12213.5
22211.4
311793.1
442862.6
542864.1
642863.4
711794.4
83.5
911791.9
102214.6
112211.5
1242863.5
132214.8
142212.3
1542862.2
1642861.3
1711792.5
1811792.4
1911791.5
2011794.8
  • digestion of miMeasure and 6 different synthetic miRNA patterns against endogenous miR122
  • gel seperation
  • gel purification
  • sequencing


12/10/2010

4 miraPCRs for binding sites of miR4286.

  • one PCR with an annealing temperature of 45 degree and 16 cycles
  • second miraPCR with annealing temperature of 57 degree and also 16 cycles
    • nucleotide removal
    • digestion
    • gel seperation
    • gel purification
    • ligation in miMeasure and pSB1C3
    • transformation

13/10/2010

  • pick colonies
  • about 8 hours later: do minipreps
  • send for sequencing

14/10/2010

  • a reasonable
  • Screening of 40 colonies of miR4286 (colony PCR)
  • Screening of 32 colonies of miR320b (colony PCR)


all negative


15/10/2010

ligation of miMeasure construct:

miR-122
miR-122ran
miR-1179
miR-221

each in 0S/0L/10S/10L versions



16/10/2010

transformation of each ligation to TOP10 E.coli

cells were plated on LB-amp plates


17/10/2010

96 colony PCRs - 8 from each plate
ALL NEGATIVE!!!!


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