Difference between revisions of "Team:Paris Saclay/Notebook/July/5"
(→B-Galactosidase and luciferase test on transformed BL21) |
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''By Caroline and Mathilde'' | ''By Caroline and Mathilde'' | ||
| − | The 04/06/2016 transformations show white colony growth for gBlocks 4.2 clone 3 and GFP1-9 | + | 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 | + | Thus this last plasmid pPS16_007 was transformed again following the same protocol as the 21/06/2016 with : |
| − | 50μg of competent DH5α cells | + | *50μg of competent DH5α cells |
| − | 4.1 ligation product in 5μL of the plasmid pUC19 | + | *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) | + | *4.1 clone 6 extracted DNA (which presented a good size strip on electrophoresis) |
6 clones of each of the other transformations [[Team:Paris_Saclay/Notebook/June/28#pPS16_001|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 [[Team:Paris_Saclay/Experiments#HeatShockCompetent|transformation]] was made with 50µL of cells and 5µL of plasmid. 50µL of transformed bacteria were plated on LB + Ampicillin (50µg/mL). | 6 clones of each of the other transformations [[Team:Paris_Saclay/Notebook/June/28#pPS16_001|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 [[Team:Paris_Saclay/Experiments#HeatShockCompetent|transformation]] was made with 50µL of cells and 5µL of plasmid. 50µL of transformed bacteria were plated on LB + Ampicillin (50µg/mL). | ||
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* 2µL of loading dye | * 2µL of loading dye | ||
* 5 µL of the digestion product | * 5 µL of the digestion product | ||
| − | * 5 µL of | + | * 5 µL of water |
| − | + | [[File:T--Paris_Saclay--160705_bringingDNAcloser_casdigestion_échelle1.JPG|400px|thumb|right|DS-NMcas, DS-SPcasN-, DS-ST1casN- and DS-TDcasN- digestion (Migration)]] | |
| − | + | ||
| − | + | ||
===Biobrick characterization=== | ===Biobrick characterization=== | ||
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|colspan="6"|K1372001 | |colspan="6"|K1372001 | ||
| − | |colspan="6"|K1372001 + | + | |colspan="6"|K1372001 + pcl_TAA |
| − | |colspan="6"|K1372001 + | + | |colspan="6"|K1372001 + pcl_TAG |
| − | |colspan="6"|K1372001 + | + | |colspan="6"|K1372001 + pcl_Tq |
|- | |- | ||
!Clone | !Clone | ||
| Line 88: | Line 86: | ||
|} | |} | ||
| − | Cultures were spun down for 5min at | + | Cultures were spun down for 5min at 13000rpm. 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 13000rpm at 4°C. |
For the luciferase test, 4mL of Luc buffer were mixed to 80μL of ATP and 8μL of luciferine. | For the luciferase test, 4mL of Luc buffer were mixed to 80μL of ATP and 8μL of luciferine. | ||
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''By Alice and Laetitia'' | ''By Alice and Laetitia'' | ||
| − | 10 µL of | + | 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. |
{{Team:Paris_Saclay/notebook_footer}} | {{Team:Paris_Saclay/notebook_footer}} | ||
Latest revision as of 12:16, 18 August 2016
