Difference between revisions of "Team:Paris Saclay/Notebook/June/28"
(→Puc19-gBlocks ligation) |
(→Interlab study) |
||
| (22 intermediate revisions by 3 users not shown) | |||
| Line 4: | Line 4: | ||
====Stock solutions preparation==== | ====Stock solutions preparation==== | ||
*200 mL '''60% glycerol''' stock: 120mL glycerol 100% + 80mL water. | *200 mL '''60% glycerol''' stock: 120mL glycerol 100% + 80mL water. | ||
| − | *200mL ''' | + | *200mL '''CH<sub>3</sub>COOK 5M''' stock: 98.15g of CH<sub>3</sub>COOK powder + 60mL water, shake and add water to 200mL |
| − | *100mL '''Solution III''' (plasmid extraction protocol): 28.5 mL water + 60mL | + | *100mL [[Team:Paris_Saclay/Experiments#solIII|'''Solution III''']] (plasmid extraction protocol): 28.5 mL water + 60mL CH<sub>3</sub>COOK 5M + 11.5 mL of pure liquid CH<sub>3</sub>COOH |
| − | *200mL '''TE''' stock: 2mL Tris+ 0.4 mL EDTA + water to 200mL | + | *200mL [[Team:Paris_Saclay/Experiments#TE|'''TE''']] stock: 2mL Tris+ 0.4 mL EDTA + water to 200mL |
| Line 13: | Line 13: | ||
''By Caroline, Alice, Lea and Charlene, with Isabelle help'' | ''By Caroline, Alice, Lea and Charlene, with Isabelle help'' | ||
| − | Cells culture created on the 27/06/2016 were | + | Cells culture created on the 27/06/2016 were analyzed with cytometer. |
Results were as expected. | Results were as expected. | ||
| Line 20: | Line 20: | ||
===Visualization=== | ===Visualization=== | ||
====pUC19 digestion==== | ====pUC19 digestion==== | ||
| − | + | pUC19 was digested by HincII. A fragment of 2.7kb (linearized plasmid) is expected. | |
| − | pUC19 was digested | + | |
| − | ====pUC19-gBlocks ligation==== | + | [[File:T--Paris_Saclay--160628_visualization_pUC19digestion_echelle.jpg|400px|thumb|right|Migration of pUC19 (digested)]] |
| + | |||
| + | ====pUC19-gBlocks ligation (cf. [[Team:Paris_Saclay/Experiments#Ligation|protocol]])==== | ||
''By Charlene'' | ''By Charlene'' | ||
| Line 69: | Line 70: | ||
| − | + | By using ligation, we hope to obtain the following plasmids: | |
{| class="wikitable" | {| class="wikitable" | ||
| Line 84: | Line 85: | ||
|- | |- | ||
!'''Plasmid name''' | !'''Plasmid name''' | ||
| − | |pPS16_001 | + | |pPS16_001<div id="pPS16_001"></div> |
| − | |pPS16_002 | + | |pPS16_002<div id="pPS16_002"></div> |
| − | |pPS16_003 | + | |pPS16_003<div id="pPS16_003"></div> |
| − | |pPS16_005 | + | |pPS16_005<div id="pPS16_005"></div> |
| − | |pPS16_006 | + | |pPS16_006<div id="pPS16_006"></div> |
| − | |pPS16_007 | + | |pPS16_007<div id="pPS16_007"></div> |
| − | |pPS16_008 | + | |pPS16_008<div id="pPS16_008"></div> |
| − | |pPS16_009 | + | |pPS16_009<div id="pPS16_009"></div> |
|} | |} | ||
| + | |||
| + | The gBlock 2.2 was not avaiblable at the time, that is why it was not transformed with the orthers. But when it will be received it will inserted in puc19 and name after pPS16_004 <div id="pPS16_004"></div>. | ||
====Transformation with ligation products==== | ====Transformation with ligation products==== | ||
''By Caroline and Charlene'' | ''By Caroline and Charlene'' | ||
| − | DH5α cells were transformed with ligation products using the | + | DH5α cells were transformed with ligation products using the [[Team:Paris_Saclay/Experiments#HeatShockCompetent|usual protocol]]. |
| − | + | Transformed cells were spread on Petri dishes with LB medium containing 50µg/mL of ampicillin and covered with 0.5µL of 80mg/mL XGal and 0.5µL of 1M IPTG. | |
| − | + | ||
===Bringing DNA closer=== | ===Bringing DNA closer=== | ||
| Line 105: | Line 107: | ||
''By Naiane and Lea'' | ''By Naiane and Lea'' | ||
| − | Plasmids from cell culture of the 27/06/2016 were extracted following the | + | Plasmids from cell culture of the 27/06/2016 were extracted following the [[Team:Paris_Saclay/Experiments#PlasmidExtraction|usual protocol]]. |
====Cells transformation==== | ====Cells transformation==== | ||
''By Naiane'' | ''By Naiane'' | ||
| − | Extracted plasmids (2µL) were transformed into DH5α competent cells (50µL) using the | + | Extracted plasmids (2µL) were transformed into DH5α competent cells (50µL) using the [[Team:Paris_Saclay/Experiments#HeatShockCompetent|usual protocol]]. |
| Line 117: | Line 119: | ||
''By Lea'' | ''By Lea'' | ||
| − | The | + | The [[Team:Paris_Saclay/Experiments#PlasmidExtraction|usual protocol]] was used to extract K1372001 from DH5α cells, except the phenol chloroform step was not done. |
| − | ===BL21 competent cell culture=== | + | ====BL21 competent cell culture==== |
''By Caroline'' | ''By Caroline'' | ||
| − | A colony of BL21 cells was put into 4mL of LB medium and incubated overnight at 37°C, 180 rpm. | + | A colony of BL21 cells was put into 4mL of LB medium and incubated overnight at 37°C, 180 rpm. For this part of the project, we will used BL21 e. coli strain because K1372001 contained a tRNAsup for UAG which DH5alp has naturally. |
{{Team:Paris_Saclay/notebook_footer}} | {{Team:Paris_Saclay/notebook_footer}} | ||
Latest revision as of 14:17, 25 July 2016
