Team:Heidelberg/Notebook/miRNA Kit/October
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- | {{:Team:Heidelberg/ | + | {{:Team:Heidelberg/Single}} |
- | {{:Team:Heidelberg/ | + | {{:Team:Heidelberg/Single_Pagetop|note_mirna_kit}} |
+ | {{:Team:Heidelberg/Side_Top}} | ||
+ | |||
+ | {| cellpadding="5" cellspacing="0" align="center" style="text-align: center; color:#009be1; border: 1.5px solid #000000;" | ||
+ | |- border="0" | ||
+ | ! colspan="7" style="background:#f09600;" | [https://2010.igem.org/Team:Heidelberg/Notebook/miRNA_Kit/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:#009be1" | ||
+ | |colspan="6"| ||'''1''' | ||
+ | |- style="background:#f2f2f2; color:#009be1" | ||
+ | |'''2'''||'''3'''||'''4'''||'''5'''||'''6'''||'''7'''||'''8''' | ||
+ | |- style="background:#f2f2f2; color:#009be1" | ||
+ | |'''9'''||'''10'''||'''11'''||'''12'''||'''13'''||'''14'''||'''15''' | ||
+ | |- style="background:#f2f2f2; color:#009be1" | ||
+ | |'''16'''||'''17'''||'''18'''||'''19'''||'''20'''||'''[https://2010.igem.org/Team:Heidelberg/Notebook/miRNA_Kit/August#21/08/2010 21]'''||'''[https://2010.igem.org/Team:Heidelberg/Notebook/miRNA_Kit/August#22.2F08.2F2010 22]''' | ||
+ | |- style="background:#f2f2f2; color:#009be1" | ||
+ | |'''[https://2010.igem.org/Team:Heidelberg/Notebook/miRNA_Kit/August#23.2F08.2F2010 23]'''||'''[https://2010.igem.org/Team:Heidelberg/Notebook/miRNA_Kit/August#24.2F08.2F2010 24]'''||'''[https://2010.igem.org/Team:Heidelberg/Notebook/miRNA_Kit/August#25.2F08.2F2010 25]'''||'''[https://2010.igem.org/Team:Heidelberg/Notebook/miRNA_Kit/August#26.2F08.2F2010 26]'''||'''[https://2010.igem.org/Team:Heidelberg/Notebook/miRNA_Kit/August#27.2F08.2F2010 27]'''||'''[https://2010.igem.org/Team:Heidelberg/Notebook/miRNA_Kit/August#28.2F08.2F2010 28]'''||'''[https://2010.igem.org/Team:Heidelberg/Notebook/miRNA_Kit/August#29.2F08.2F2010 29]''' | ||
+ | |- style="background:#f2f2f2; color:#009be1" | ||
+ | |'''[[Igem2010/Main/synthetic_miR_Kit/August#30/08/2010|30]]'''||'''[[Igem2010/Main/synthetic_miR_Kit/August#31/08/2010|31]]'''||colspan="5"| | ||
+ | |} | ||
+ | |||
+ | |||
+ | {| cellpadding="5" cellspacing="0" align="center" style="text-align: center; color:#78b41e; border: 1.5px solid #333333;" | ||
+ | |- border="0" | ||
+ | ! colspan="7" style="background:#009be1;" | [https://2010.igem.org/Team:Heidelberg/Notebook/miRNA_Kit/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:#78b41e" | ||
+ | |colspan="2"| ||'''[https://2010.igem.org/Team:Heidelberg/Notebook/miRNA_Kit/September#01.2F09.2F2010 1]'''||'''[https://2010.igem.org/Team:Heidelberg/Notebook/miRNA_Kit/September#02.2F09.2F2010 2]'''||'''[https://2010.igem.org/Team:Heidelberg/Notebook/miRNA_Kit/September#03.2F09.2F2010 3]'''||'''[https://2010.igem.org/Team:Heidelberg/Notebook/miRNA_Kit/September#04.2F09.2F2010 4]'''||'''[https://2010.igem.org/Team:Heidelberg/Notebook/miRNA_Kit/September#05.2F09.2F2010 5]''' | ||
+ | |- style="background:#f2f2f2; color:#78b41e" | ||
+ | |'''[https://2010.igem.org/Team:Heidelberg/Notebook/miRNA_Kit/September#06.2F09.2F2010 6]'''||'''[https://2010.igem.org/Team:Heidelberg/Notebook/miRNA_Kit/September#07.2F09.2F2010 7]'''||'''[https://2010.igem.org/Team:Heidelberg/Notebook/miRNA_Kit/September#08.2F09.2F2010 8]'''||'''[https://2010.igem.org/Team:Heidelberg/Notebook/miRNA_Kit/September#09.2F09.2F2010 9]'''||'''[https://2010.igem.org/Team:Heidelberg/Notebook/miRNA_Kit/September#10.2F09.2F2010 10]'''||'''[https://2010.igem.org/Team:Heidelberg/Notebook/miRNA_Kit/September#11.2F09.2F2010 11]'''||'''[https://2010.igem.org/Team:Heidelberg/Notebook/miRNA_Kit/September#12.2F09.2F2010 12]''' | ||
+ | |- style="background:#f2f2f2; color:#78b41e" | ||
+ | |'''[https://2010.igem.org/Team:Heidelberg/Notebook/miRNA_Kit/September#13.2F09.2F2010 13]'''||'''[https://2010.igem.org/Team:Heidelberg/Notebook/miRNA_Kit/September#14.2F09.2F2010 14]'''||'''[https://2010.igem.org/Team:Heidelberg/Notebook/miRNA_Kit/September#15.2F09.2F2010 15]'''||'''[https://2010.igem.org/Team:Heidelberg/Notebook/miRNA_Kit/September#16.2F09.2F2010 16]'''||'''[https://2010.igem.org/Team:Heidelberg/Notebook/miRNA_Kit/September#17.2F09.2F2010 17]'''||'''[https://2010.igem.org/Team:Heidelberg/Notebook/miRNA_Kit/September#18.2F09.2F2010 18]'''||'''[https://2010.igem.org/Team:Heidelberg/Notebook/miRNA_Kit/September#19.2F09.2F2010 19]''' | ||
+ | |- style="background:#f2f2f2; color:#78b41e" | ||
+ | |'''[https://2010.igem.org/Team:Heidelberg/Notebook/miRNA_Kit/September#20.2F09.2F2010 20]'''||'''[https://2010.igem.org/Team:Heidelberg/Notebook/miRNA_Kit/September#21.2F09.2F2010 21]'''||'''[https://2010.igem.org/Team:Heidelberg/Notebook/miRNA_Kit/September#22.2F09.2F2010 22]'''||'''[https://2010.igem.org/Team:Heidelberg/Notebook/miRNA_Kit/September#23.2F09.2F2010 23]'''||'''[https://2010.igem.org/Team:Heidelberg/Notebook/miRNA_Kit/September#24.2F09.2F2010 24]'''||'''[https://2010.igem.org/Team:Heidelberg/Notebook/miRNA_Kit/September#25.2F09.2F2010 25]'''||'''[https://2010.igem.org/Team:Heidelberg/Notebook/miRNA_Kit/September#26.2F09.2F2010 26]''' | ||
+ | |- style="background:#f2f2f2; color:#78b41e" | ||
+ | |'''[https://2010.igem.org/Team:Heidelberg/Notebook/miRNA_Kit/September#27.2F09.2F2010 27]'''||'''[https://2010.igem.org/Team:Heidelberg/Notebook/miRNA_Kit/September#28.2F09.2F2010 28]'''||'''[https://2010.igem.org/Team:Heidelberg/Notebook/miRNA_Kit/September#29.2F09.2F2010 29]'''||'''[https://2010.igem.org/Team:Heidelberg/Notebook/miRNA_Kit/September#22.2F30.2F2010 30]'''||colspan="3"| | ||
+ | |- style="background:#f2f2f2; color:#78b41e" | ||
+ | | 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/miRNA_Kit/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:#78b41e" | ||
+ | |colspan="4"| ||'''[https://2010.igem.org/Team:Heidelberg/Notebook/miRNA_Kit/October#01.2F10.2F2010 1]'''||'''[https://2010.igem.org/Team:Heidelberg/Notebook/miRNA_Kit/October#02.2F10.2F2010 2]'''||'''[https://2010.igem.org/Team:Heidelberg/Notebook/miRNA_Kit/October#03.2F10.2F2010 3]''' | ||
+ | |- style="background:#f2f2f2; color:#f09600" | ||
+ | |'''[https://2010.igem.org/Team:Heidelberg/Notebook/miRNA_Kit/October#04.2F10.2F2010 4]'''||'''[https://2010.igem.org/Team:Heidelberg/Notebook/miRNA_Kit/October#05.2F10.2F2010 5]''' | ||
+ | |'''[https://2010.igem.org/Team:Heidelberg/Notebook/miRNA_Kit/October#06.2F10.2F2010 6]'''||'''[https://2010.igem.org/Team:Heidelberg/Notebook/miRNA_Kit/October#07.2F10.2F2010 7]'''||'''[https://2010.igem.org/Team:Heidelberg/Notebook/miRNA_Kit/October#08.2F10.2F2010 8]'''||'''[https://2010.igem.org/Team:Heidelberg/Notebook/miRNA_Kit/October#09.2F10.2F2010 9]'''||'''[https://2010.igem.org/Team:Heidelberg/Notebook/miRNA_Kit/October#10.2F10.2F2010 10]''' | ||
+ | |- style="background:#f2f2f2; color:#f09600" | ||
+ | |'''[https://2010.igem.org/Team:Heidelberg/Notebook/miRNA_Kit/October#11.2F10.2F2010 11]'''||'''[https://2010.igem.org/Team:Heidelberg/Notebook/miRNA_Kit/October#12.2F10.2F2010 12]'''|'''[https://2010.igem.org/Team:Heidelberg/Notebook/miRNA_Kit/October#13.2F10.2F2010 13]'''||'''[https://2010.igem.org/Team:Heidelberg/Notebook/miRNA_Kit/October#14.2F10.2F2010 14]'''||'''[https://2010.igem.org/Team:Heidelberg/Notebook/miRNA_Kit/October#15.2F09.2F2010 15]'''||'''[https://2010.igem.org/Team:Heidelberg/Notebook/miRNA_Kit/October#16.2F10.2F2010 16]'''||'''[https://2010.igem.org/Team:Heidelberg/Notebook/miRNA_Kit/October#17.2F10.2F2010 17]'''||'''[https://2010.igem.org/Team:Heidelberg/Notebook/miRNA_Kit/October#18.2F10.2F2010 18]''' | ||
+ | |- style="background:#f2f2f2; color:#f09600" | ||
+ | |'''[https://2010.igem.org/Team:Heidelberg/Notebook/miRNA_Kit/October#19/10/2010 19]'''||'''20'''||'''21'''||'''22'''||'''23'''||'''24'''||'''25''' | ||
+ | |- style="background:#f2f2f2; color:#f09600" | ||
+ | |'''26'''||'''27'''||'''28'''||'''29'''||'''30'''||colspan="3"| | ||
+ | |- style="background:#f2f2f2; color:#f09600" | ||
+ | | colspan="7"| <span style="color:#ffffff">-</span> | ||
+ | |} | ||
+ | |||
+ | |||
+ | {{:Team:Heidelberg/Side_Bottom}} | ||
+ | |||
+ | __NOTOC__ | ||
+ | |||
+ | |||
===01/10/2010=== | ===01/10/2010=== | ||
[[Image:20101001_tetR_verdauEPanno.jpg|thumb|350px|right|Gel 101001-1. Digestion of miniprep no. 8, 9, 14 and 16 with EcoRI and PstI. Odd lanes are undigested controls, even lanes are digested minis]] | [[Image:20101001_tetR_verdauEPanno.jpg|thumb|350px|right|Gel 101001-1. Digestion of miniprep no. 8, 9, 14 and 16 with EcoRI and PstI. Odd lanes are undigested controls, even lanes are digested minis]] | ||
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repressor construct: | repressor construct: | ||
* colony 8, 9, 14 and 16 were chosen for miniprep. They consist of the complete TetR construct in pBS 1C3 | * colony 8, 9, 14 and 16 were chosen for miniprep. They consist of the complete TetR construct in pBS 1C3 | ||
- | * digestion of the minis with EcoRI and PstI for cloning into | + | * digestion of the minis with EcoRI and PstI for cloning into [http://partsregistry.org/Part:pSB1A3 pSB1A3], SAP of [http://partsregistry.org/Part:pSB1A3 pSB1A3], digestion was tested on gel and proven positive (see Gel 101001-1) |
* nucleotide removal, then ligation of TetR8 and TetR14 equimolar with SAPed backbone, TetR9 and TetR16 2:1 with not-SAPed backbone, transformation with TOP10 cells (10µl on 50µl ''E. coli'') | * nucleotide removal, then ligation of TetR8 and TetR14 equimolar with SAPed backbone, TetR9 and TetR16 2:1 with not-SAPed backbone, transformation with TOP10 cells (10µl on 50µl ''E. coli'') | ||
<br /> | <br /> | ||
Line 26: | Line 94: | ||
repressor construct:<br/> | repressor construct:<br/> | ||
- | * [[Igem2010/Main/Protocols/Colony_PCR | colony-PCR]] of 6 colonies for each plate of | + | * [[Igem2010/Main/Protocols/Colony_PCR | colony-PCR]] of 6 colonies for each plate of previous day transformations |
** analysis, see gel 101002-1 | ** analysis, see gel 101002-1 | ||
* twice test digestion of promising mini-preps, following [https://2010.igem.org/3A_Assembly standard protocol recommendations] <br/> | * twice test digestion of promising mini-preps, following [https://2010.igem.org/3A_Assembly standard protocol recommendations] <br/> | ||
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===04/10/2010=== | ===04/10/2010=== | ||
repressor construct:<br/> | repressor construct:<br/> | ||
- | * [[Igem2010/Main/Protocols/Colony_PCR | colony-PCR]] of 6 colonies for each plate of | + | * [[Igem2010/Main/Protocols/Colony_PCR | colony-PCR]] of 6 colonies for each plate of previous day transformations |
** gel 101004-1 reveals almost positive samples | ** gel 101004-1 reveals almost positive samples | ||
* submission of each a promising mini-prep for sequencing of binding site and correct insert | * submission of each a promising mini-prep for sequencing of binding site and correct insert | ||
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===05/10/2010=== | ===05/10/2010=== | ||
repressor construct:<br/> | repressor construct:<br/> | ||
- | * <i>sequencing results</i> from | + | * <i>sequencing results</i> from previous day: |
** once or twice the binding site in each of the T1 constructs, respectively | ** once or twice the binding site in each of the T1 constructs, respectively | ||
** once the binding site for the T2 construct | ** once the binding site for the T2 construct | ||
*** re-trial of ligation for two binding sites in T2 | *** re-trial of ligation for two binding sites in T2 | ||
- | * planned transfection into HeLa and HUH cells (non-liver and liver to prove ON-targeting) in ratio 6:1 = repressor:operator(tuning) as recommended in the [http:// | + | * planned transfection into HeLa and HUH cells (non-liver and liver to prove ON-targeting) in ratio 6:1 = repressor:operator(tuning) as recommended in the [http://www.roche-applied-science.com/pack-insert/1815091a.pdf manual] |
new approach: | new approach: | ||
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===06/10/2010=== | ===06/10/2010=== | ||
- | * waiting for colonies from | + | * waiting for colonies from previous night, which did not grow after all |
===07/10/2010=== | ===07/10/2010=== | ||
* repetition of shhAAT cloning | * repetition of shhAAT cloning | ||
* touch-down PCR setup: 8.5 µl ddH<sub>2</sub>O, 0.5 µl template DNA (pcDNA5, 50 ng/µl), 0.5 µl each primer (i.e. [[Igem2010/Main/Primer_database#Standart_Kit_Cloning_Primers | D7 & D16]], 10 µl Phusion PCR MasterMix (2x) | * touch-down PCR setup: 8.5 µl ddH<sub>2</sub>O, 0.5 µl template DNA (pcDNA5, 50 ng/µl), 0.5 µl each primer (i.e. [[Igem2010/Main/Primer_database#Standart_Kit_Cloning_Primers | D7 & D16]], 10 µl Phusion PCR MasterMix (2x) | ||
- | * touch-down PCR protocol: [ | + | * touch-down PCR protocol: [https://2010.igem.org/Team:Heidelberg/Notebook/Methods#Plasmid-PCR like this] but with increase of annealing temperature from 68°C to 61°C (i. e. 0.2°C per cycle) |
* go on like [[Igem2010/Main/synthetic miR Kit/October#05/10/2010 | 05/10/2010]] but growth on freshly prepared plates | * go on like [[Igem2010/Main/synthetic miR Kit/October#05/10/2010 | 05/10/2010]] but growth on freshly prepared plates | ||
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===09/10/2010=== | ===09/10/2010=== | ||
- | * sequencing | + | * sequencing the day before was fine but construct contains TetO<sub>2</sub>, thus: maybe Q2 and Q3 were confused |
** that would explain why measurements with TetR constructs are not working properly even though sequences were fine | ** that would explain why measurements with TetR constructs are not working properly even though sequences were fine | ||
* cloning of binding sites for shhAAT (perfect binding site generated via annealing, and randomized ones PCR amplified) | * cloning of binding sites for shhAAT (perfect binding site generated via annealing, and randomized ones PCR amplified) | ||
** touch-down PCR setup: 17 µl ddH<sub>2</sub>O, 4 µl each oligo (i.e. respective forward primer + second strand oligo[[Igem2010/Main/Primer_database#Standart_Kit_Cloning_Primers | D121]]), 25 µl Phusion PCR MasterMix (2x) | ** touch-down PCR setup: 17 µl ddH<sub>2</sub>O, 4 µl each oligo (i.e. respective forward primer + second strand oligo[[Igem2010/Main/Primer_database#Standart_Kit_Cloning_Primers | D121]]), 25 µl Phusion PCR MasterMix (2x) | ||
- | *** touch-down PCR protocol: [ | + | *** touch-down PCR protocol: [https://2010.igem.org/Team:Heidelberg/Notebook/Methods#shRNA-PCR_protocol like this] but with increase of annealing temperature from 70°C to 62°C (i. e. 0.5°C per cycle, 16x) and four additional cycles with 62°C |
** annealing of perfect binding against shhAAT sites with an oligo-based strategy following the protocol from the [[Igem2010/Main/synthetic miR Kit/October#03/10/2010 | 3<sup>rd</sup> October]] | ** annealing of perfect binding against shhAAT sites with an oligo-based strategy following the protocol from the [[Igem2010/Main/synthetic miR Kit/October#03/10/2010 | 3<sup>rd</sup> October]] | ||
** digestion of PCR amplified binding sites with AgeI and XhoI, digestion of Q2 backbone containing the shhAAT with XhoI and XmaI, afterwards heat inactivation, nucleotide removal and ligation following [https://2010.igem.org/3A_Assembly standard protocol recommendations] | ** digestion of PCR amplified binding sites with AgeI and XhoI, digestion of Q2 backbone containing the shhAAT with XhoI and XmaI, afterwards heat inactivation, nucleotide removal and ligation following [https://2010.igem.org/3A_Assembly standard protocol recommendations] | ||
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===10/10/2010=== | ===10/10/2010=== | ||
[[Image:20101010 colonypcr shhAAT p 12 22 bs LOLA.png|thumb|350px|right|Gel 101010-1. Colony PCR for construct containing shhAAT and binding sites. Expected is an amplified fragment a bit smaller than 300 bp which can be seen on lane 3, 4, 6, 7, 8 (perfect binding sites), 12, 13, 14, 16 and 17 (randomized binding sites). Lane 1 and Lane 26: 1kb Plus Ladder (Invitrogen)]] | [[Image:20101010 colonypcr shhAAT p 12 22 bs LOLA.png|thumb|350px|right|Gel 101010-1. Colony PCR for construct containing shhAAT and binding sites. Expected is an amplified fragment a bit smaller than 300 bp which can be seen on lane 3, 4, 6, 7, 8 (perfect binding sites), 12, 13, 14, 16 and 17 (randomized binding sites). Lane 1 and Lane 26: 1kb Plus Ladder (Invitrogen)]] | ||
- | * colony pcr reveals some positive clones (see gel 101010-1) from | + | * colony pcr reveals some positive clones (see gel 101010-1) from previous day |
** inoculation of 5 ml LB Amp cultures for mini-prep over night | ** inoculation of 5 ml LB Amp cultures for mini-prep over night | ||
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In presence of Tet repressor and miR122 that is not targeting it, less of firefly luciferase is expressed, but when miR sAg is expressed is recovered to 60% of original activity. | In presence of Tet repressor and miR122 that is not targeting it, less of firefly luciferase is expressed, but when miR sAg is expressed is recovered to 60% of original activity. | ||
+ | <br /><br /> | ||
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+ | <br /><br /> | ||
===11/10/2010=== | ===11/10/2010=== | ||
- | * mini-prep of ten cultures from | + | * mini-prep of ten cultures from previous day and direct transfection to test the system |
** amount: each 100 µl (c=2.5 ng/µl) | ** amount: each 100 µl (c=2.5 ng/µl) | ||
<br/> | <br/> | ||
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*** 25 µl Phusion PCR MasterMix (2x) | *** 25 µl Phusion PCR MasterMix (2x) | ||
** touch-down PCR protocol: | ** touch-down PCR protocol: | ||
- | *** [ | + | *** [https://2010.igem.org/Team:Heidelberg/Notebook/Methods#shRNA-PCR_protocol like this] but with increase of annealing temperature from 70°C to 62°C (i. e. 0.5°C per cycle, 16x) |
*** and four additional cycles with 62°C, elongation time only 10 seconds | *** and four additional cycles with 62°C, elongation time only 10 seconds | ||
* digestion of generated binding sites and the pSMB_miMeasure backbone with EcoRI and PstI, then nucleotide removal | * digestion of generated binding sites and the pSMB_miMeasure backbone with EcoRI and PstI, then nucleotide removal | ||
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*** elongation time: 90<nowiki>''</nowiki> | *** elongation time: 90<nowiki>''</nowiki> | ||
** <u>no positive</u> results, maybe due to insufficient screening primers (one for BBb standard, the other one for CMV resulting in an 1300 bp fragment) | ** <u>no positive</u> results, maybe due to insufficient screening primers (one for BBb standard, the other one for CMV resulting in an 1300 bp fragment) | ||
- | *** as a consequence: repetition of entire cloning with [http://www.fermentas.com/templates/files/tiny_mce/coa_pdf/coa_ef0511.pdf SAP] treated backbone | + | *** as a consequence: repetition of entire cloning with [http://www.fermentas.com/templates/files/tiny_mce/coa_pdf/coa_ef0511.pdf SAP] treated backbone following day |
===13/10/2010=== | ===13/10/2010=== | ||
measurement construct for single binding sites: <br/> | measurement construct for single binding sites: <br/> | ||
- | * repetition of cloning from | + | * repetition of cloning from 11/10/2010 |
* <u>note:</u> vector concentration of pSMB_miMeasure is okay but after digestion and nucleotide removal it is always pretty low | * <u>note:</u> vector concentration of pSMB_miMeasure is okay but after digestion and nucleotide removal it is always pretty low | ||
** (loss of 75% of DNA on average whereas other DNA yields to 90% of purified DNA as compared to digested amount) | ** (loss of 75% of DNA on average whereas other DNA yields to 90% of purified DNA as compared to digested amount) | ||
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===18/10/2010=== | ===18/10/2010=== | ||
- | * [ | + | * [https://2010.igem.org/Team:Heidelberg/Notebook/Methods#Colony_PCR colony-PCR] of 4 colonies for each plate of [[Igem2010/Main/synthetic miR Kit/October#17/10/2010 | previous day transformations]] using reverse binding site oligo and forward hAAT primer expecting a fragment round about 1300 bp |
** gel 101018-1 reveals almost positive samples except two (data not yet shown) | ** gel 101018-1 reveals almost positive samples except two (data not yet shown) | ||
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* dilution and co-transfection with miRsAg expressing construct into HeLa cells for ELISA measurements | * dilution and co-transfection with miRsAg expressing construct into HeLa cells for ELISA measurements | ||
- | + | ===23/10/2010=== | |
- | + | seeding HeLa cells | |
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- | + | ===24/10/2010=== | |
- | + | [https://2010.igem.org/Team:Heidelberg/Notebook/Methods#Transfection Transfection] | |
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- | + | 1) HeLa luc2-miR122BS and miR122 | |
- | + | 2) for ELISA - pBS_U6 containing hAAT with miRsAg imperfect binding sites, and miRsAg | |
- | + | ratios 1:1 ans 4:1 | |
- | + | ||
- | + | ===26/10/2010=== | |
- | + | * [https://2010.igem.org/Team:Heidelberg/Notebook/Methods#Dual_Luciferase_Assay Dual Luciferase Assay] | |
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- | + | [[Image:PsiCheck.png|thumb|center|600px|'''Figure 3: Tuning of gene expression through different imperfect miR122 binding sites in psiCHECK-2.''' Construct was transfected into HeLa cells together with an plasmid expressing miR122. Control without binding site was used for normalization.]] | |
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- | + | [[Image:Haat U6HD2010.jpg|thumb|center|600px|'''Figure 1: Tuning of gene expression through different imperfect shRNA miR binding sites in pBS_U6''' Gene expression quantified via dual luciferase assay for constructs containing different imperfect binding sites for shhAAT.]] | |
- | + | [[Image:Haat_H1HD2010.jpg|thumb|center|600px|'''Figure 2: Tuning of gene expression through different imperfect shRNA miR binding sites in pBS_H1.''' Gene expression quantified via dual luciferase assay for constructs containing different imperfect binding sites for shhAAT.]] | |
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- | + | * [https://2010.igem.org/Team:Heidelberg/Notebook/Methods#ELISA ELISA] was performed on media in which HeLa cells we grown, as hAAT is secreted by cells. | |
- | {{:Team:Heidelberg/ | + | {{:Team:Heidelberg/Single_Bottom}} |
Latest revision as of 09:58, 27 October 2010
01/10/2010repressor construct:
02/10/2010
03/10/2010repressor construct:
04/10/2010repressor construct:
05/10/2010repressor construct:
new approach:
06/10/2010
07/10/2010
08/10/2010
09/10/2010
10/10/2010
Tuning construct in which luc2 expression is driven by CMV promoter with TetO2 also expressing either miR122 or miR sAg was cotransfected with Tet repressor under regulation of perfect miR sAg binding site. Amounts transfected were: 2.5ng of tuning construct, 20ng of miRNA expressing plamid (either miR122 or miR sAg) and 5ng of repressor construct. To keep transfection efficiency the same we used sheared salmon sperm DNA as a stuffer.
11/10/2010
measurement construct for single binding sites:
12/10/2010measurement construct for single binding sites:
13/10/2010measurement construct for single binding sites:
14/10/2010
15/10/2010
16/10/2010
17/10/2010
18/10/2010
19/10/2010
23/10/2010seeding HeLa cells 24/10/20101) HeLa luc2-miR122BS and miR122 2) for ELISA - pBS_U6 containing hAAT with miRsAg imperfect binding sites, and miRsAg ratios 1:1 ans 4:1 26/10/2010
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