Difference between revisions of "Team:Paris Saclay/Notebook/June/28"
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| − | {{Team: Paris_Saclay/notebook_header}} | + | {{Team:Paris_Saclay/notebook_header}} |
| − | =Tuesday | + | =Tuesday 28<sup>th</sup> June= |
==Lab work== | ==Lab work== | ||
====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 19: | Line 19: | ||
===Visualization=== | ===Visualization=== | ||
| − | ==== | + | ====pUC19 digestion==== |
| − | + | pUC19 was digested by HincII. A fragment of 2.7kb (linearized plasmid) is expected. | |
| − | + | ||
| − | ==== | + | [[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'' | ||
GBlocks were resuspended into 100µL of TE buffer (except for 4-1 which was resuspended into 50µL of TE) in order to get a concentration of 10ng/mL after a quick spin. | GBlocks were resuspended into 100µL of TE buffer (except for 4-1 which was resuspended into 50µL of TE) in order to get a concentration of 10ng/mL after a quick spin. | ||
Solutions were vortexed, incubated for 20min at 50°C, vortexed another time and quickly spun down. | Solutions were vortexed, incubated for 20min at 50°C, vortexed another time and quickly spun down. | ||
| + | The ratio vector size/insert size x 3.5 is calculated to determine if insert is enough in excess compared to vector (it has to be superior than 7). 3.5 represents the ratio insert concentration/ vector concentration. | ||
{| class="wikitable" | {| class="wikitable" | ||
| Line 62: | Line 64: | ||
|} | |} | ||
| − | 3 | + | 3 conditions were tested (2 controls): |
| − | # 1µL digested vector + 9µL | + | # 1µL digested vector + 9µL water |
| − | # 1µL digested vector + 1µL ligase + 1µL ligation buffer + 7µL | + | # 1µL digested vector + 1µL ligase + 1µL ligation buffer + 7µL water |
# 1µL digested vector + 1µL ligase + 1µL ligation buffer + 7µL insert | # 1µL digested vector + 1µL ligase + 1µL ligation buffer + 7µL insert | ||
| + | |||
| + | |||
| + | By using ligation, we hope to obtain the following plasmids: | ||
| + | |||
| + | {| class="wikitable" | ||
| + | |- | ||
| + | !'''gBlock''' | ||
| + | !1-1 | ||
| + | !1-2 | ||
| + | !2-1 | ||
| + | !3-1 | ||
| + | !3-2 | ||
| + | !4-1 | ||
| + | !4-2 | ||
| + | !GFP1-9 | ||
| + | |- | ||
| + | !'''Plasmid name''' | ||
| + | |pPS16_001<div id="pPS16_001"></div> | ||
| + | |pPS16_002<div id="pPS16_002"></div> | ||
| + | |pPS16_003<div id="pPS16_003"></div> | ||
| + | |pPS16_005<div id="pPS16_005"></div> | ||
| + | |pPS16_006<div id="pPS16_006"></div> | ||
| + | |pPS16_007<div id="pPS16_007"></div> | ||
| + | |pPS16_008<div id="pPS16_008"></div> | ||
| + | |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 78: | 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 90: | 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'' | ||
| − | |||
| − | {{Team: Paris_Saclay/notebook_footer}} | + | 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}} | ||
Latest revision as of 14:17, 25 July 2016
