Difference between revisions of "Team:Paris Saclay/Notebook/June/29"
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{{Team:Paris_Saclay/notebook_header}} | {{Team:Paris_Saclay/notebook_header}} | ||
=Wednesday 29<sup>th</sup> June= | =Wednesday 29<sup>th</sup> June= | ||
| − | |||
===Visualization=== | ===Visualization=== | ||
| − | ====Culture of clones | + | ====Culture of clones containing the gBlocks ==== |
''By Lea and Marion'' | ''By Lea and Marion'' | ||
| − | 6 clones of each construction were selected and grown overnight in 3.5mL of LB (50µg/ml Amp) at 37°C, 180rpm. | + | 6 clones of each construction were selected (transformed cells with the plasmid containing the gBlocks are supposed to grow white on xGal/IPTG) and grown overnight in 3.5mL of LB (50µg/ml Amp) at 37°C, 180rpm. |
===BioBrick characterization=== | ===BioBrick characterization=== | ||
| − | ====BL21 electro-competent cells preparation==== | + | ====BL21 electro-competent cells [[Team:Paris_Saclay/Experiments#ElectroCompetent|preparation and transformation]]==== |
''By Charlene'' | ''By Charlene'' | ||
| − | 200µL of BL21 pre-culture was added to 15mL of LB and incubated for 4h at 37°C. When | + | 200µL of BL21 pre-culture was added to 15mL of LB and incubated for 4h at 37°C. When OD<sub>600nm</sub>=0.68, cells were centrifuged for 10 minutes at 4000rpm and washed twice with addition of 10mL glycerol (10%) followed by 10 minutes of centrifugation at 4000rpm. Cells were mixed with 200µL glycerol and kept -80°C. |
| − | = | + | {| class="wikitable" |
| − | + | |- | |
| + | !Plasmids used | ||
| + | !Description | ||
| + | !Use | ||
| + | |- | ||
| + | |pcl_TAA | ||
| + | |Promoteur_RBS_LacZ_TAA_Luciferase_Terminator | ||
| + | |Negative control = base level | ||
| + | |- | ||
| + | |pcl_TAG | ||
| + | |Promoteur_RBS_LacZ_TAG_Luciferase_Terminator | ||
| + | |Our interest plasmids allowing us to measured the biobrick activity | ||
| + | |- | ||
| + | |pcl_Tq | ||
| + | |Promoteur_RBS_LacZ_CodonSens_Luciferase_Terminator | ||
| + | |Positive control = luciferase 100% activity | ||
| + | |} | ||
We realized four transformations with the following plasmids: | We realized four transformations with the following plasmids: | ||
| Line 26: | Line 41: | ||
50µL of cells were added in the cuvettes with 1µL DNA (diluted 1/10). Everything was done at 4°C. | 50µL of cells were added in the cuvettes with 1µL DNA (diluted 1/10). Everything was done at 4°C. | ||
| − | Transformation did not work for K1372001 and K1372001+pcl_TAA because the cuvettes were broken | + | Transformation did not work for K1372001 and K1372001+pcl_TAA because the cuvettes were broken (electroporation time constant was equal to 1.6ms. Cells died during transformation. |
| − | Transformation worked for K1372001+pcl_TAG and K1372001+pcl_Tq | + | Transformation worked for K1372001+pcl_TAG and K1372001+pcl_Tq (time constant was equal to 6ms. We added 1mL of LB to the cells and incubated them for 1h at 37°C. |
The following cells were plated on Petri dishes (30µg/mL Cm + 50µg/mL streptomycin): | The following cells were plated on Petri dishes (30µg/mL Cm + 50µg/mL streptomycin): | ||
| Line 38: | Line 53: | ||
''By Naiane'' | ''By Naiane'' | ||
| − | K1372001 plasmid was digested with BglI using the following quantities: 3 µL DNA, 2µL buffer (O), 1 µL enzyme (BglI), 14µL water. The mix was incubated for 1h at 37°C and 10 min at 55°C and kept at -20°C until the gel electrophoresis was done. Fragments are expected to size 1.2kb and 2.4kb. | + | K1372001 plasmid was [[Team:Paris_Saclay/Experiments#PlasmidDigestion|digested with BglI]] using the following quantities: 3 µL DNA, 2µL buffer (O), 1 µL enzyme (BglI), 14µL water. The mix was incubated for 1h at 37°C and 10 min at 55°C and kept at -20°C until the gel electrophoresis was done. Fragments are expected to size 1.2kb and 2.4kb. |
====BL21 cell culture==== | ====BL21 cell culture==== | ||
| − | We added 200µL of the BL21 culture made on 28/06 in 15ml of LB and incubated it overnight at 37°C, 180rpm. | + | We added 200µL of the BL21 culture made on 28/06 in 15ml of LB and incubated it overnight at 37°C, 180rpm. We need a new BL21 culture to redo the experiment because two of the transformations (K1372001 and pcl_TAA) did not work out (broken electroporation cuvettes). |
===Bringing DNA closer=== | ===Bringing DNA closer=== | ||
| Line 47: | Line 62: | ||
''By Naiane'' | ''By Naiane'' | ||
| − | Plasmids received from Addgene were digested with AvrII. | + | Plasmids received from Addgene were [[Team:Paris_Saclay/Experiments#PlasmidDigestion|digested with AvrII]]. |
{| class="wikitable" | {| class="wikitable" | ||
| Line 72: | Line 87: | ||
!Plasmid name | !Plasmid name | ||
!Plasmid size (kb) | !Plasmid size (kb) | ||
| − | ! | + | !Expected digestion product size (kb) |
|- | |- | ||
|DS-NMcas | |DS-NMcas | ||
| Line 91: | Line 106: | ||
|} | |} | ||
| + | The migration's result was: | ||
| + | |||
| + | [[File:T--Paris_Saclay--160629_bringingDNAcloser_casdigestion_échelle.JPG|200px|thumb|left|TD,NM,SP,ST1 electrophoresis]] | ||
{{Team:Paris_Saclay/notebook_footer}} | {{Team:Paris_Saclay/notebook_footer}} | ||
Latest revision as of 13:24, 9 October 2016
