Team:Heidelberg/Notebook/miRNA Kit/September
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===01/09/2010=== | ===01/09/2010=== | ||
- | [[Image:20100901_gel1bb.png|thumb|350 px| | + | [[Image:20100901_gel1bb.png|thumb|350 px|left|Gel 100901-1]] |
- | [[Image:20100901_gel2bb.png|thumb|350 px| | + | [[Image:20100901_gel2bb.png|thumb|350 px|left|Gel 100901-2]] |
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* 8 colonies of each plate (see [[Igem2010/Main/synthetic miR Kit/August#31/08/2010 | previous day]]) were picked and colony-PCR was performed. Furthermore, the colonies were used for inocculation of LB miniprep cultures. The numbers behind the construct name refer to vector type (1 = not SAP treated, 2 = dephosphorylated using SAP; see previous day). For hRluc (1300 bp band)), Luc2 (2300 bp band), CMV_TetO (1000 bp band) and the FRT (500 bp band) site, positive colonies could be observed (gel 100901-1 and 100901-2). | * 8 colonies of each plate (see [[Igem2010/Main/synthetic miR Kit/August#31/08/2010 | previous day]]) were picked and colony-PCR was performed. Furthermore, the colonies were used for inocculation of LB miniprep cultures. The numbers behind the construct name refer to vector type (1 = not SAP treated, 2 = dephosphorylated using SAP; see previous day). For hRluc (1300 bp band)), Luc2 (2300 bp band), CMV_TetO (1000 bp band) and the FRT (500 bp band) site, positive colonies could be observed (gel 100901-1 and 100901-2). | ||
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===02/09/2010=== | ===02/09/2010=== | ||
- | [[Image:20100802_gel1.png|thumb|350 px| | + | [[Image:20100802_gel1.png|thumb|350 px|left|Gel 100902-1]] |
+ | [[Image:20100902_gel2.png|thumb|350 px|right|Gel 100902-2]] | ||
+ | [[Image:20100902_gel3.png|thumb|350 px|right|Gel 100902-3]] | ||
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* 8 colonies were picked from plates 1 and 4 colonies from plates 2 from the previous days cloning and analyzed via colony PCR performed according to the [https://2010.igem.org/Team:Heidelberg/Notebook/Methods#Colony_PCR PCR standard protocol]. The results of the colony PCR were analyzed on a 1 % agarose gel run for 1 h @ 100 V (gel 100902-1 and 100902-2). The following colonies were afterwards inocculated (LB miniprep cultures): | * 8 colonies were picked from plates 1 and 4 colonies from plates 2 from the previous days cloning and analyzed via colony PCR performed according to the [https://2010.igem.org/Team:Heidelberg/Notebook/Methods#Colony_PCR PCR standard protocol]. The results of the colony PCR were analyzed on a 1 % agarose gel run for 1 h @ 100 V (gel 100902-1 and 100902-2). The following colonies were afterwards inocculated (LB miniprep cultures): | ||
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:* shRNA9 (1.2) | :* shRNA9 (1.2) | ||
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* The cultures inocculated the day before were mini prepped by applying a Qiagen Miniprep Kit. Afterwards, a test digestion with NotI was performed and the result was analyzed on a 1 % agarose gel, run for 35 min @ 100 V (gel 100902-3). According to the test digestion result, the following samples were send for sequencing @ GATC: | * The cultures inocculated the day before were mini prepped by applying a Qiagen Miniprep Kit. Afterwards, a test digestion with NotI was performed and the result was analyzed on a 1 % agarose gel, run for 35 min @ 100 V (gel 100902-3). According to the test digestion result, the following samples were send for sequencing @ GATC: | ||
:* CMV_TetO2 (.4, 1.6) | :* CMV_TetO2 (.4, 1.6) | ||
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:* hRluc (1.1, 1.3, 1.4) | :* hRluc (1.1, 1.3, 1.4) | ||
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* cloning of shRNAs into pcDNA5/FRT/TO | * cloning of shRNAs into pcDNA5/FRT/TO | ||
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===04/09/2010=== | ===04/09/2010=== | ||
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* ligation of both inserts for the tuning construct, extraction of the right insert (3000bp) and ligation into the backbone | * ligation of both inserts for the tuning construct, extraction of the right insert (3000bp) and ligation into the backbone | ||
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===09/09/2010=== | ===09/09/2010=== | ||
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** PCR of CMV shRNA which leads to a size of 1200bp | ** PCR of CMV shRNA which leads to a size of 1200bp | ||
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===11/09/2010=== | ===11/09/2010=== | ||
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[[Image:Rb100911_testdigestshRNA.jpg|thumb|350px|right|test digest]] | [[Image:Rb100911_testdigestshRNA.jpg|thumb|350px|right|test digest]] | ||
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===12/09/2010=== | ===12/09/2010=== | ||
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* miniprep of shRNA constructs and Tuning constructs | * miniprep of shRNA constructs and Tuning constructs | ||
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* Colony- PCR for the cloning products done on the previous day (gels 100914-1 and 100914-2 on the right) | * Colony- PCR for the cloning products done on the previous day (gels 100914-1 and 100914-2 on the right) | ||
* positive clones were Mini-Prepped and test digested | * positive clones were Mini-Prepped and test digested | ||
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===15/09/2010=== | ===15/09/2010=== | ||
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* Selection of colonies from the plates (see previous days' cloning) via colony-PCR using the standard sequencing primers; a ~5.5 kb fragment should be visible for the assembled construct K1-K8, if colony-PCR was positive (gel 100917-1 and 100917-2). No sample shows a 5.5 kb band, but that's most likely due to the very difficult amplification of such a long fragment with Fermentas 2x PCR MasterMix (Taq-based). Therefor 3 miniprep cultures were inocculated for each construct (K1-K8). The TetR oligo insertion and the Kozag_hRluc_BGH cloning into pSB1C3 gave the right bands on the gel. | * Selection of colonies from the plates (see previous days' cloning) via colony-PCR using the standard sequencing primers; a ~5.5 kb fragment should be visible for the assembled construct K1-K8, if colony-PCR was positive (gel 100917-1 and 100917-2). No sample shows a 5.5 kb band, but that's most likely due to the very difficult amplification of such a long fragment with Fermentas 2x PCR MasterMix (Taq-based). Therefor 3 miniprep cultures were inocculated for each construct (K1-K8). The TetR oligo insertion and the Kozag_hRluc_BGH cloning into pSB1C3 gave the right bands on the gel. | ||
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===19/09/2010=== | ===19/09/2010=== | ||
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[[Image:Promoter_test_220910_hd2010.jpg| thumb | 600px | centre | Promoter strength characterization in HEK 293 T-REx cell line]] | [[Image:Promoter_test_220910_hd2010.jpg| thumb | 600px | centre | Promoter strength characterization in HEK 293 T-REx cell line]] | ||
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+ | [[Image:23092010 DLA promoters cell lines.jpg | thumb | 600px | centre| Promoter strength characterization in different cell lines]] | ||
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===23/09/2010=== | ===23/09/2010=== | ||
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[https://2010.igem.org/Team:Heidelberg/Notebook/Methods#Dual_Luciferase_Assay Dual Luciferase Assay] was performed as on [[https://2010.igem.org/Team:Heidelberg/Notebook/miRNA_Kit/September#22.2F09.2F2010 previous day]]. Cells were not harvested after 20h, but 48h. | [https://2010.igem.org/Team:Heidelberg/Notebook/Methods#Dual_Luciferase_Assay Dual Luciferase Assay] was performed as on [[https://2010.igem.org/Team:Heidelberg/Notebook/miRNA_Kit/September#22.2F09.2F2010 previous day]]. Cells were not harvested after 20h, but 48h. | ||
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===24/09/2010=== | ===24/09/2010=== | ||
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** ligate 150ng and 250ng of insert into 30ng [http://partsregistry.org/Part:pSB1A3 pSB1A3] 1h at RT and transform ligation | ** ligate 150ng and 250ng of insert into 30ng [http://partsregistry.org/Part:pSB1A3 pSB1A3] 1h at RT and transform ligation | ||
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===27/09/2010=== | ===27/09/2010=== | ||
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===29/09/2010=== | ===29/09/2010=== | ||
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* ligation of P56 (14) with R8 and R11 and SAPed backbone and control ligation with backbone only, transformation | * ligation of P56 (14) with R8 and R11 and SAPed backbone and control ligation with backbone only, transformation | ||
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Latest revision as of 05:57, 27 October 2010
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All Ligations were controlled by loading 10 ul of ligation reactiong onto a 1 % agarose gel (100907-1), run for 35 min @ 100 V
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Subsequent Transformation into DH5alpha cells
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Each PCR was performed in two replicates. The result of the PCRs was analyzed on a 1 % agarose gel (100915-1), run for 1 h @ 100 V. The PCR nr. 2 was subsequently PCR purified by applying a Qiagen PCR purification KIT and used for the cloning 16/09/2010
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28/09/2010tuning construct:
29/09/2010tuning construct:
repressor construct:
30/09/2010tuning construct:
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