Team:Heidelberg/Notebook/BSDesign/July
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{{:Team:Heidelberg/Single_Pagetop|note_BSDesign}} | {{:Team:Heidelberg/Single_Pagetop|note_BSDesign}} | ||
{{:Team:Heidelberg/Side_Top}} | {{:Team:Heidelberg/Side_Top}} | ||
+ | |||
+ | {| cellpadding="5" cellspacing="0" align="center" style="text-align: center; color:#4e93a4; border:1.53px solid #333333;" | ||
+ | |- border="0" | ||
+ | ! colspan="7" style="background:#c85000;" | [https://2010.igem.org/Team:Heidelberg/Notebook/BSDesign/July<font color="white">July</font>] | ||
+ | |- 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''' | ||
+ | |- style="background:#f2f2f2; color:#f09600" | ||
+ | |colspan="3"| ||'''1'''||'''2'''||'''3'''||'''4''' | ||
+ | |- style="background:#f2f2f2; color:#f09600" | ||
+ | |'''[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]''' | ||
+ | |- style="background:#f2f2f2; color:#f09600" | ||
+ | |'''[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]''' | ||
+ | |- style="background:#f2f2f2; color:#f09600" | ||
+ | |'''[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''' | ||
+ | |- style="background:#f2f2f2; color:#f09600" | ||
+ | |'''[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]'''|| | ||
+ | |- style="background:#f2f2f2; color:#f09600" | ||
+ | | colspan="7"| | ||
+ | <span style="color:#ffffff">-</span> | ||
+ | |} | ||
+ | |||
+ | |||
+ | {| cellpadding="5" cellspacing="0" align="center" style="text-align: center; color:#f09600; border: 1.5px solid #000000;" | ||
+ | |- border="0" | ||
+ | ! colspan="7" style="background:#f09600;" | [https://2010.igem.org/Team:Heidelberg/Notebook/BSDesign/August<font color="white">August</font>] | ||
+ | |- 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''' | ||
+ | |- style="background:#f2f2f2; color:#f09600" | ||
+ | |colspan="6"| ||'''[https://2010.igem.org/Team:Heidelberg/Notebook/BSDesign/August#01.2F08.2F2010 1]''' | ||
+ | |- 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]''' | ||
+ | |- style="background:#f2f2f2; color:#f09600" | ||
+ | |'''[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''' | ||
+ | |- style="background:#f2f2f2; color:#f09600" | ||
+ | |'''16'''||'''17'''||'''18'''||'''19'''||'''20'''||'''21'''||'''22''' | ||
+ | |- style="background:#f2f2f2; color:#f09600" | ||
+ | |'''23'''||'''24'''||'''25'''||'''26'''||'''27'''||'''28'''||'''29''' | ||
+ | |- style="background:#f2f2f2; color:#f09600" | ||
+ | |'''30'''||'''31'''||colspan="5"| | ||
+ | |} | ||
+ | |||
+ | |||
+ | {| cellpadding="5" cellspacing="0" align="center" style="text-align: center; color:#f09600; border: 1.5px solid #333333;" | ||
+ | |- border="0" | ||
+ | ! colspan="7" style="background:#009be1;" | [https://2010.igem.org/Team:Heidelberg/Notebook/BSDesign/September<font color="#ffecba">September</font>] | ||
+ | |- 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''' | ||
+ | |- style="background:#f2f2f2; color:#f09600" | ||
+ | |colspan="2"| ||'''1'''||'''2'''||'''3'''||'''4'''||'''5''' | ||
+ | |- style="background:#f2f2f2; color:#f09600" | ||
+ | |'''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]''' | ||
+ | |- 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" | ||
+ | |'''[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]''' | ||
+ | |- style="background:#f2f2f2; color:#f09600" | ||
+ | |'''[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"| | ||
+ | |- style="background:#f2f2f2; color:#f09600" | ||
+ | |colspan="7"| | ||
+ | <span style="color:#ffffff">-</span> | ||
+ | |} | ||
+ | |||
+ | |||
+ | {| cellpadding="5" cellspacing="0" align="center" style="text-align: center; color:#f09600; border: 1.5px solid #333333;" | ||
+ | |- border="0" | ||
+ | ! colspan="7" style="background:#78b41e;" | [https://2010.igem.org/Team:Heidelberg/Notebook/BSDesign/October<font color="white">October</font>] | ||
+ | |- 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''' | ||
+ | |- style="background:#f2f2f2; color:#f09600" | ||
+ | |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]''' | ||
+ | |- style="background:#f2f2f2; color:#f09600" | ||
+ | |'''[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]''' | ||
+ | |- style="background:#f2f2f2; color:#f09600" | ||
+ | |'''[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]''' | ||
+ | |- style="background:#f2f2f2; color:#f09600" | ||
+ | |'''[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''' | ||
+ | |- style="background:#f2f2f2; color:#f09600" | ||
+ | |'''25'''||'''26'''||'''27'''||'''28'''||'''29'''||'''30'''||'''31''' | ||
+ | |- style="background:#f2f2f2; color:#f09600" | ||
+ | |colspan="7"| | ||
+ | <span style="color:#ffffff">-</span> | ||
+ | |} | ||
{{:Team:Heidelberg/Side_Bottom}} | {{:Team:Heidelberg/Side_Bottom}} | ||
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== 05/07/2010 - 11/07/2010 == | == 05/07/2010 - 11/07/2010 == | ||
<br /> | <br /> | ||
- | === Preperation of competent E. coli Top10 and DH5alpha === | + | === Preperation of chemically competent cells E. coli Top10 and DH5alpha === |
* plating of E. coli Top10 and DH5alpha on a agar plate (LB, without Amp); Preperation of competent cells according to the following protocol: | * plating of E. coli Top10 and DH5alpha on a agar plate (LB, without Amp); Preperation of competent cells according to the following protocol: | ||
First, a 20 ml over night culture was inoculated in antibiotic free LB medium from a fresh single colony and transferred into 400 ml antibiotic free LB medium the next day. This culture was incubated at 37 °C while shacking until an OD600 of 0.5 – 0.6 was achieved. The culture was than cooled down on ice, centrifuged (8 min, 4 °C, 3500 rpm), the supernatant discarded and the pellet resuspended in 10 ml 100 mM CaCl2. After addition of further 190 ml 100 mM CaCl2 the suspension was incubated on ice for 30 min. The suspension was than again centrifuged (8 min, 4 °C, 3500 rpm), the supernatant discarded, the pellet resuspended in 20 ml 82.5 mM CaCl2 with 17.5 % glycerol and aliquoted. The aliquots were flash frozen in liquid nitrogen and than stored at -80 °C until usage. | First, a 20 ml over night culture was inoculated in antibiotic free LB medium from a fresh single colony and transferred into 400 ml antibiotic free LB medium the next day. This culture was incubated at 37 °C while shacking until an OD600 of 0.5 – 0.6 was achieved. The culture was than cooled down on ice, centrifuged (8 min, 4 °C, 3500 rpm), the supernatant discarded and the pellet resuspended in 10 ml 100 mM CaCl2. After addition of further 190 ml 100 mM CaCl2 the suspension was incubated on ice for 30 min. The suspension was than again centrifuged (8 min, 4 °C, 3500 rpm), the supernatant discarded, the pellet resuspended in 20 ml 82.5 mM CaCl2 with 17.5 % glycerol and aliquoted. The aliquots were flash frozen in liquid nitrogen and than stored at -80 °C until usage. | ||
<br /> | <br /> | ||
+ | |||
=== Cell Culture Starting === | === Cell Culture Starting === | ||
* thawing of Huh-7, HeLa p4 and HEK-293 cells according to the following protocol | * thawing of Huh-7, HeLa p4 and HEK-293 cells according to the following protocol | ||
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:* once, the probe was nearly completely thawn, cells were thrown into pre-warmed DMEM (10 % FCS, L-Glut, P/S) and gentely mixed | :* once, the probe was nearly completely thawn, cells were thrown into pre-warmed DMEM (10 % FCS, L-Glut, P/S) and gentely mixed | ||
:* cells were spinned down at 800 rpm, 3 min; supernatant was discarded | :* cells were spinned down at 800 rpm, 3 min; supernatant was discarded | ||
- | :* the | + | :* the pellet was resuspended in 10 ml DMEM an plated on a p100 cell culture dish in the following media according to the different cell lines: |
<br /> | <br /> | ||
Media for Hela and Hek cells: | Media for Hela and Hek cells: | ||
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=== design of measurement standard === | === design of measurement standard === | ||
<br /> | <br /> | ||
- | * in order to design an appropriety standard respecting all the rules according to the standard of mammalian synthetic biology (RFC12), we developed a dual reporter | + | * in order to design an appropriety standard respecting all the rules according to the standard of mammalian synthetic biology (RFC12), we developed a dual reporter measurement construct with the following properties: |
:* bidirectional CMV promoter driving both reporter genes | :* bidirectional CMV promoter driving both reporter genes | ||
:* distabilized EGFP and EBFP2 as reporter genes, as they are really similar in their whole structure and amino-acid sequence | :* distabilized EGFP and EBFP2 as reporter genes, as they are really similar in their whole structure and amino-acid sequence |
Latest revision as of 03:17, 28 October 2010
Binding Site Design - July05/07/2010 - 11/07/2010
Preperation of chemically competent cells E. coli Top10 and DH5alpha
First, a 20 ml over night culture was inoculated in antibiotic free LB medium from a fresh single colony and transferred into 400 ml antibiotic free LB medium the next day. This culture was incubated at 37 °C while shacking until an OD600 of 0.5 – 0.6 was achieved. The culture was than cooled down on ice, centrifuged (8 min, 4 °C, 3500 rpm), the supernatant discarded and the pellet resuspended in 10 ml 100 mM CaCl2. After addition of further 190 ml 100 mM CaCl2 the suspension was incubated on ice for 30 min. The suspension was than again centrifuged (8 min, 4 °C, 3500 rpm), the supernatant discarded, the pellet resuspended in 20 ml 82.5 mM CaCl2 with 17.5 % glycerol and aliquoted. The aliquots were flash frozen in liquid nitrogen and than stored at -80 °C until usage.
Cell Culture Starting
Media for HUH-7 cells:
12/07/2010 - 19/07/2010
Preperation of RNA extracts for miRNA profiling
Protocol
design of diraPCR oligos
design of measurement standard
please, find more information about the measurement standard design in the measurment standard page
20/07/2010
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21/07/2010
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22/07/2010
23/07/2010
26/07/2010
single binding site synthesis
PCR protocol:
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27/07/2010
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Single Binding Site Synthesis - Optimization
28/07/2010
raPCR for the construction of anti miRsAg-shRNA sponges
31/07/2010
diraPCR for constructing hsa-886-3p binding site patterns
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