Toggle navigation Home Team Team Media Collaborations Sponsors Acknowledgements Project Background Design CRISPR/Cas9 Strategy Experiments Notebook Results Perspective Interlab Study Parts Parts Basic Parts Composite Parts Human Pratices Overview Societal Issues of CRISPR/Cas9 Responsible Research and Innovation GMO regulation Integrated Practices Engagement Model Attributions Safety Contents 1 Tuesday 5th July 1.1 Lab work 1.1.1 Visualization 1.1.1.1 Transformation of DH5α|pPS16_004 and DH5α|pPS16_007 1.1.1.2 Plasmid extraction 1.1.2 Bringing DNA closer 1.1.2.1 Gel migration of DS-NMcas, DS-SPcasN-, DS-ST1casN- and DS-TDcasN- digestion 1.1.3 Biobrick characterization 1.1.3.1 B-Galactosidase and luciferase test on transformed BL21 1.1.3.2 Biobrick K1372001 : clone 1 and clone 2 plamsid digestion 1.1.3.3 Biobrick K1372001 : clone 1 and clone 2 culture Tuesday 5th July Lab work Visualization Transformation of DH5α|pPS16_004 and DH5α|pPS16_007 By Caroline and Mathilde The 04/06/2016 transformations show white colony growth for gBlocks 4.2 (pPS16_008) clone 3 and GFP1-9 (pPS16_009) clone 1, but blue colonies only for gBlocks 4.1 (pPS16_007) clone 3. Thus this last plasmid pPS16_007 was transformed again following the same protocol as the 21/06/2016 with : 50μg of competent DH5α cells 4.1 ligation product in 5μL of the plasmid pUC19 or 4.1 clone 6 extracted DNA (which presented a good size strip on electrophoresis) 6 clones of each of the other transformations pPS16_001, pPS16_002, pPS16_003, pPS16_005 and pPS16_006 were grown in 4mL of LB with Ampicillin (50µg/mL) at 37°C, 180rpm. For pPS16_002 only 2 clones were cultivated because there was not more colony. To obtain more colonies, another transformation was made with 50µL of cells and 5µL of plasmid. 50µL of transformed bacteria were plated on LB + Ampicillin (50µg/mL). Plasmid extraction By Laetitia and Alice The usual protocol was used to extract plasmids pPS16_008 from 250μL of clone 3 overnight culture and pPS16_009 from 320μL of clone 1 overnight culture (from 28/06/16 transformation). Plasmids were resuspended in 10μL of water with RNAse (50μg/mL). Bringing DNA closer Gel migration of DS-NMcas, DS-SPcasN-, DS-ST1casN- and DS-TDcasN- digestion By Naiane 2µL of loading dye 5 µL of the digestion product 5 µL of water DS-NMcas, DS-SPcasN-, DS-ST1casN- and DS-TDcasN- digestion (Migration) Biobrick characterization B-Galactosidase and luciferase test on transformed BL21 By Charlene Cultures tested Plasmid(s) K1372001 K1372001 + pcl_TAA K1372001 + pcl_TAG K1372001 + pcl_Tq Clone 1 2 1 2 1 2 1 2 1 2 1 2 1 2 1 2 1 2 1 2 1 2 1 2 Salicylate concentration 0 30µM 1mM 0 30µM 1mM 0 30µM 1mM 0 30µM 1mM Cultures were spun down for 5min at 1300rpm. Supernatant was discarded and glass marbles previously cleaned with 1M nitric acid were added with 50μL of Luc buffer. Tubes were vortexed for 30min at 4°C. Everything was then done on ice. 150μL of Luc bufer was added and cells were centrifuged for 15min at 13000rom at 4°C. For the luciferase test, 4mL of Luc buffer were mixed to 80μL of ATP and 8μL of luciferine. For the βGal test, 400μL of Z buffer mixed with 10μL of extract and 100μL of ONPG were incubated for 8min at 30°C. Then 250μL of STOP buffer was added. OD420nm was measured. Ratio between luciferase activity and β-Gal activity Biobrick K1372001 : clone 1 and clone 2 plamsid digestion By Mathilde The same protocol as the 04.07.2016 was followed. But the incubation step was accidentally interrupted so the epxerience has to conducted again. Biobrick K1372001 : clone 1 and clone 2 culture By Alice and Laetitia 10 µL of cultures of the two clones expressing K1372001 were put in culture in 3mL of LB + 3µL of chloramphenicol. Cultures were incubated at 37°C, 180 rpm.
By Caroline and Mathilde
The 04/06/2016 transformations show white colony growth for gBlocks 4.2 (pPS16_008) clone 3 and GFP1-9 (pPS16_009) clone 1, but blue colonies only for gBlocks 4.1 (pPS16_007) clone 3.
Thus this last plasmid pPS16_007 was transformed again following the same protocol as the 21/06/2016 with :
6 clones of each of the other transformations pPS16_001, pPS16_002, pPS16_003, pPS16_005 and pPS16_006 were grown in 4mL of LB with Ampicillin (50µg/mL) at 37°C, 180rpm. For pPS16_002 only 2 clones were cultivated because there was not more colony. To obtain more colonies, another transformation was made with 50µL of cells and 5µL of plasmid. 50µL of transformed bacteria were plated on LB + Ampicillin (50µg/mL).
By Laetitia and Alice
The usual protocol was used to extract plasmids pPS16_008 from 250μL of clone 3 overnight culture and pPS16_009 from 320μL of clone 1 overnight culture (from 28/06/16 transformation). Plasmids were resuspended in 10μL of water with RNAse (50μg/mL).
By Naiane
By Charlene
Cultures tested
Cultures were spun down for 5min at 1300rpm. Supernatant was discarded and glass marbles previously cleaned with 1M nitric acid were added with 50μL of Luc buffer. Tubes were vortexed for 30min at 4°C. Everything was then done on ice. 150μL of Luc bufer was added and cells were centrifuged for 15min at 13000rom at 4°C.
For the luciferase test, 4mL of Luc buffer were mixed to 80μL of ATP and 8μL of luciferine. For the βGal test, 400μL of Z buffer mixed with 10μL of extract and 100μL of ONPG were incubated for 8min at 30°C. Then 250μL of STOP buffer was added. OD420nm was measured.
By Mathilde
The same protocol as the 04.07.2016 was followed. But the incubation step was accidentally interrupted so the epxerience has to conducted again.
By Alice and Laetitia
10 µL of cultures of the two clones expressing K1372001 were put in culture in 3mL of LB + 3µL of chloramphenicol. Cultures were incubated at 37°C, 180 rpm.
